Â

ultrafiltration
handbook
Tools and Techniques for
Life Science Research
Introduction
This handbook is written for research scientists who are using, or who are interested in using,
ultrafiltration (UF) technology in their work.

Our goal is to provide researchers with a handy reference piece that will allow them to optimize
their UF separations and provide them with purified and/or concentrated sample that is ready for
downstream experimentation.

The intention is not to make ultrafiltration experts out of research scientists—in fact, one of the
appealing benefits of the technology is its simplicity. However, having a basic understanding of
how ultrafiltration works, and how it can be applied to everyday research, enables the researcher
to select the right membrane or device for their particular experiment.

The information contained in this handbook is culled from decades of collective experience of
Millipore and Amicon applications scientists and technical service employees, as well as the
company’s library of technical publications on ultrafiltration, UF devices, and UF applications.
Millipore customers have also contributed data, helping to make this handbook a complete
resource for ultrafiltration.

Ultrafiltration has evolved significantly since it was first introduced in the 1970s, and it will
continue to evolve as life science research progresses. New device platforms and
new applications work have expanded the use of UF into new areas, such as genomics,
proteomics, drug compound screening, and small molecule assays.

To keep up with the latest developments, visit our web site: www.millipore.com.

The Protein Research Team

Millipore Corporation
Life Sciences Division
Table of Contents
Overview of Membrane Filtration
M em b r a n e P rocesses . . . . . . . . . . . . . 2
Ultrafiltration . . . . . . . . . . . . . . . . . . . . . .2
Microfiltration . . . . . . . . . . . . . . . . . . . . .2
Reverse Osmosis . . . . . . . . . . . . . . . . . . .4
R e c o ver y . . . . . . . . . . . . . . . . . . . . . . . . 4
Nominal Molecular Weight Limit . . . . . . .4
Nucleotide Cut-off . . . . . . . . . . . . . . . . . .4
Retention . . . . . . . . . . . . . . . . . . . . . . . .5
Concentration Polarization . . . . . . . . . . . .5
Flux . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Effects of Operating Parameters on Flux . . .6
Importance of Recovery . . . . . . . . . . . . . .6
Mode of Operation . . . . . . . . . . . . . . 7
Normal vs. Tangential Flow Filtration . . . . .7
Diafiltration . . . . . . . . . . . . . . . . . . . . . 7
Dialysis vs. Diafiltration . . . . . . . . . . . . . .8
Guide to Selecting Membranes
and Devices
Membrane Selection . . . . . . . . . . . . . 9
Types of Membranes . . . . . . . . . . . . . . . .9
Membranes Organized by
Typical Recoveries . . . . . . . . . . . . . . . .9
Membranes Organized by Application . .10
Device Selection . . . . . . . . . . . . . . . . 10
Small Volume Devices . . . . . . . . . . . . . .12
Medium Volume Devices . . . . . . . . . . . .13
Large Volume and TFF Devices . . . . . . . .14
Stirred Cells . . . . . . . . . . . . . . . . . . . . .14
Specialty Devices and Kits . . . . . . . . . . .15
Protocols
P ro tein s . . . . . . . . . . . . . . . . . . . . . . . . 16
Concentration, Desalting, and
Buffer Exchange with Amicon Ultra
or Microcon Centrifugal Filters . . . . . . .16
Detergent Removal with Microcon,
Amicon Ultra or Centricon
Centrifugal Filters . . . . . . . . . . . . . . . .20
Two-Dimensional Electrophoresis
Sample Preparation with
Amicon Ultra Centrifugal Filters . . . . . .22
Purification of Serum Peptides
for Biomarker Research with
Amicon Ultra Centrifugal Filters . . . . . .24
Rapid Antibody Concentration with
Amicon Ultra Centrifugal Filters . . . . . .27
Removal of Unincorporated Label from
Labeled Protein with Amicon Ultra
or Centricon Centrifugal Filters . . . . . .28
Urine Concentration with
Amicon Ultra Centrifugal Filters . . . . . .30
Affinity Purification with Ultrafree-MC
Centrifugal Filters . . . . . . . . . . . . . . . .32
Use of Centrifugal Filter Devices
as an Alternative to Stirred Cells . . . . .35
Nucleic Acids . . . . . . . . . . . . . . . . . . . 36
Concentrating and Desalting DNA or
RNA with Microcon or Centricon
Centrifugal Filters . . . . . . . . . . . . . . . .36
Preparing Samples for Forensics
Identification Analysis with
Microcon Centrifugal Filters . . . . . . . . .39
PCR Purification with Montage PCR
Filter Units . . . . . . . . . . . . . . . . . . . . .40
Preparation of Fluorescent DNA Probe
from Human mRNA or Total RNA
with Microcon Centrifugal Filters . . . . .42
Purification of In Vitro Synthesized mRNA
with Microcon or Centricon
Centrifugal Filters . . . . . . . . . . . . . . . .44
Quantitative Recoveries of Nanogram
Amounts of Nucleic Acids with
Microcon Centrifugal Filters . . . . . . . .46
Effect of Centrifugal Ultrafiltration
on Large Fragment DNA Integrity . . . . .48
DNA Extraction from Agarose Gels
with Montage Gel Extraction Kit or
Ultrafree-DA Centrifugal Filters . . . . . .50
RNA Purification with Microcon or
Centricon Centrifugal Filters . . . . . . . .52
Enzyme Removal with Micropure-EZ
Centrifugal Filters . . . . . . . . . . . . . . . .55
H igh Th rou gh pu t Applicat ion s . . . . 58
Using MultiScreen Filter Plates with
Ultracel-10 Ultrafiltration Membrane . .58
Appendix
Glossary . . . . . . . . . . . . . . . . . . . . . . .59
1
Overview of Membrane Filtration

Membrane Processes Ultrafiltration is typically used to separate

proteins from buffer components for buffer
Ultrafiltration
exchange, desalting, or concentration. Ultrafilters
Ultrafiltration (UF) is the process of separating
are also ideal for removal or exchange of sugars,
extremely small particles and dissolved molecules
non-aqueous solvents, the separation of free from
from fluids. The primary basis for separation is
protein-bound ligands, the removal of materials of
molecular size, although in all filtration applications,
low molecular weight, or the rapid change of ionic
the permeability of a filter medium can be affected
and/or pH environment (see Figure 1.6, page 8).
by the chemical, molecular or electrostatic proper-
Depending on the protein to be retained, the most
ties of the sample. Ultrafiltration can only separate
frequently used membranes have a nominal molecular
molecules which differ by at least an order of
weight limit (NMWL) of 3 kDa to 100 kDa.
magnitude in size. Molecules of similar size can
Ultrafiltration is far gentler to solutes than
not be separated by ultrafiltration (see Figure 1.3).
processes such as precipitation. UF is more efficient
Materials ranging in size from 1K to 1000K
because it can simultaneously concentrate and
molecular weight (MW) are retained by certain
desalt solutes. It does not require a phase change,
ultrafiltration membranes, while salts and water will
which often denatures labile species, and UF can
pass through. Colloidal and particulate matter can
be performed either at room temperature or in a
also be retained. Ultrafiltration membranes can be
cold room.
used both to purify material passing through the
filter and also to collect material retained by the Microfiltration
filter. Materials significantly smaller than the pore Microfiltration (MF) is the process of removing
size rating pass through the filter and can be particles or biological entities in the 0.025 µm to
depyrogenated, clarified and separated from 10.0 µm range from fluids by passage through a
high molecular weight contaminants. Materials microporous medium such as a membrane filter.
larger than the pore size rating are retained by the Although micron-sized particles can be removed by
filter and can be concentrated or separated from use of non-membrane or depth materials such as
low molecular weight contaminants.

Reverse Osmosis Ultrafiltration Microfiltration Clarification

0.0001 µm 0.001 µm 0.01 µm 0.1 µm 1 µm 10 µm 100 µm

0.2 kDa 200 kDa 20,000 kDa

Red Blood Smallest

Sugars Proteins B. diminuta
Cell Visible Particle

Amino Acids
Carbon Black Yeast Pollens
Nucleotides

Oligo- Polio
Salts Mycoplasma E. coli Clouds Fog
nucleotides Virus

Human
Antibiotics Mammalian Virus Bacteria
Hair

Figure 1.1 Comparison of ultrafiltration with other commonly used membrane separation techniques.

2
those found in fibrous media, only a membrane filter
having a precisely defined pore size can ensure
quantitative retention. Membrane filters can be used
for final filtration or prefiltration, whereas a depth
filter is generally used in clarifying applications
where quantitative retention is not required or as a
prefilter to prolong the life of a downstream
membrane. Membrane and depth filters offer certain
advantages and limitations. They can complement
each other when used together in a microfiltration
process system or fabricated device.
The retention boundary defined by a membrane
filter can also be used as an analytical tool to
validate the integrity and efficiency of a system.
For example, in addition to clarifying or sterilizing
filtration, fluids containing bacteria can be filtered
to trap the microorganisms on the membrane surface
for subsequent culture and analysis. Microfiltration
can also be used in sample preparation to remove
intact cells and some cell debris from the lysate.
Membrane pore size cut-offs used for this type of
separation are typically in the range of 0.05 µm
to 1.0 µm.

Figure 1.2 Ultrafiltration membranes vs. traditional microporous membranes

Ultrafiltration membranes generally
have two distinct layers: a thin
(0.1– 1.5 µm), dense skin with a
pore diameter of 10 – 400 Å and
a more porous substructure. Any
species capable of passing through
the pores of the skin (whose size is
precisely controlled in manufacture)
can therefore freely pass the
Cross-section of ultrafiltration membrane.
membrane with skin and porous
substructure.
Microporous membranes are
generally rigid, continuous meshes
of polymeric material with defined
pore sizes. They are used to retain
bacteria, colloids and particulates.
Species are either retained on the
membrane surface or trapped
in its substructure.
Cross-section of traditional micro-
porous membrane with uniform
pore structure from top to bottom.

Figure 1.3 Fractionating samples using ultrafiltration membranes

A popular misconception is that one can pass a mixture of macromole-
cules through an ultrafiltration membrane and separate all molecules
whose molecular weights are larger than the membrane’s cut-off from
those whose molecular weights are smaller than the cut-off.
This expectation is based on a misunderstanding of the principles
of membrane filtration. Most scientists believe that molecules smaller
than the membrane rating will pass freely through to the filtrate while
molecules larger than the membrane rating will be quantitatively
retained.
In fact, membrane ratings are based on retention and not on
passage properties. The relative retention/passage properties for
a 100K NMWL rated membrane are illustrated below. These data
were generated using Millipore’s mixed dextran rejection procedure.
This protocol provides a more detailed look at membrane retention
1.0
0.9
Rejection Coefficient
properties than the traditional evaluation with two or three protein
species. The mixed dextran protocol uses a total of 11 dextran
standards of known molecular weight (180 to 1750K daltons) and the
relative retention/passage of the mixture is determined after filtration
by analyzing the retentate and filtrate fractions using gel permeation
chromatography.
An examination of the curve for the Ultracel PL 300K NMWL
membrane confirms that molecules larger that the 100K NMWL rating
are retained at a level greater than 90%. However, molecules smaller
than the membrane rating do not exhibit free passage to the filtrate;
for example, a 75K kDa species is retained at a 83% level and a
30K kDa species is 32% retained on this membrane.
These smaller molecules are being retained by a membrane with
a higher retention rating because the retention/passage phenomenon
is a function of both the membrane and the solution being processed.
Buffer composition as well as solute concentration can have major
impacts on the retention/passage of molecules. For example, dilute

solutions of single solutes less than the NMWL ratings are more likely
Ultracel PL 300K
0.8
NMWL membrane
to appear in the filtrate than the same molecule in a more concen-
trated solution or in a complex mixture.
0.7
However, it may be possible to affect this type of separation
0.6
between two macromolecules if there is at least 10X difference in their
molecular weight and if there is an ultrafiltration membrane available
0.5 with a NMWL rating between the molecular weights of the two
10,000 100,000 1,000,000
proteins. These separations are also generally more successful if the
Molecular Weight (kDa)
protein solution is dilute and the separation is run at low pressure.
Mixed Dextran Rejection Profile

3
 Reverse Osmosis
Reverse osmosis (RO) separates salts and small
molecules from low molecular weight solutes
(typically less than 100 daltons) at relatively
high pressures using membranes with NMWLs of
1 kDa or lower. RO membranes are normally rated
by their retention of sodium chloride while ultrafiltra-
tion membranes are characterized according to the
molecular weight of retained solutes. Millipore
water purification systems employ both reverse
osmosis membranes as well as ultrafiltration
membranes. Reverse osmosis systems are primarily
used to purify tap water to purities that exceed
distilled water quality. Ultrafiltration systems ensure
that ultrapure water is free from endotoxins as well
as nucleases for critical biological research.
Recovery
The ultimate aim of ultrafiltration is to maximize
recovery of solutes of interest, but there are many
membrane characteristics that affect that goal.
Factors affecting recovery include:
• Nominal molecular weight limit (NMWL)/
nucleotide cut-off (NCO)
• Retention
• Concentration polarization
• Flux
Nominal Molecular Weight Limit
A microfiltration membrane’s pore size rating,
typically given as a micron value, indicates that
particles larger than the rating will be retained.
Ultrafiltration membranes are rated according to
the nominal molecular weight limit (NMWL), also
sometimes referred to as molecular weight cut-off
(MWCO). The NMWL indicates that most dissolved
macromolecules with molecular weights higher than
the NMWL will be retained.
An ultrafiltration membrane with a stated NMWL
should retain (reject) at least 90% of a globular
solute of that molecular weight in daltons. However,
for a wider safety margin, the selected cut-off
should be well below the molecular weight of the
solute to be retained. When solutes are to be
exchanged, the cut-off should be substantially
above that of the passing solute. A lower NMWL
increases rejection but decreases the filtration rate
for the same membrane material.
Retention and product recovery are a function
of a variety of other factors, including the molecular
shape and size of the molecule; electrical charac-
4
teristics; sample concentration and composition;
operating conditions; and device or system
configuration. Two membranes may have the same
NMWL but will exhibit different retention of
molecules within a relatively narrow range of sizes.
In addition, slender, linear molecules (e.g., nucleic
acids) may find their way through pores that will
retain a globular species of the same weight.
Retention can also be affected by hydration with
counter-ions. Nevertheless, NMWL has proven to
be an effective general indicator of membrane
performance for globular proteins.
When using membrane ultrafiltration for sample
concentration or desalting, care must be taken to
select a membrane (or device) with a NMWL
appropriate for the application. Because there are
several considerations in determining whether a
given solute will or will not be retained by a
membrane of a specific cut-off, it is best to choose
a device with cut-off at about one half of the
molecular weight of the protein to be concentrated.
This maximizes protein recovery and minimizes
filtration time.
Nucleotide Cut-off (NCO)
For most membranes, the NMWL is determined
experimentally under a standard set of operating
conditions. These analyses typically employ purified
globular proteins to serve as markers or indicators of
the retention characteristics of an ultrafiltration
membrane. Although this approach is useful for
choosing the appropriate NMWL for most protein
research applications, selection of a membrane with
an appropriate NMWL membrane for nucleic acid
or polysaccharide purification is considerably more
complex. By virtue of the rod-like three-dimensional
structures of these molecules, these types of
molecules require a tighter membrane (with a
smaller cut-off) than do globular proteins of the
same molecular weight. It is therefore convenient
to consider the membrane retention characteristics
of nucleic acids as being related to their length
(in nucleotides) rather than their molecular weight.
Complicating matters even further are several
additional factors that affect the recovery of nucleic
acid fragments from a membrane of a given
NMWL. These factors include: the strandedness
of the DNA or RNA molecule; whether the DNA is
linear, relaxed or supercoiled (for plasmid); the ionic
strength of the solvent; the velocity of the process
stream over the membrane; and the nature of the
driving force. The overall effect is that optimal
nucleic acid recovery is achieved in low salt buffers
run under conditions of relatively low velocity (e.g.,
low vacuum pressure or low g-force). The membrane
and protocol developed for the Montage® PCR
centrifugal filter device takes into account these
conditions in order to provide for high recovery of
small PCR products (e.g., ~150 base pairs [bp]) as
quickly as possible. For purifications that are driven
by vacuum, significantly tighter membranes are
typically required to obtain optimal recovery.
If the DNA sample is in the presence of high salt
(or the device is run at a higher-than-recommended
g-force), a significantly reduced DNA recovery may
be observed. Under these conditions, higher DNA
or RNA recovery can be achieved by using a tighter
membrane. However, it will take significantly longer
to complete the purification. For applications such
as PCR where removal of unincorporated single-
stranded primers from double-stranded DNA
fragments is required, the molecular weights of
the primer and DNA fragment should differ by at
least an order of magnitude for efficient separation.
Millipore offers devices that are specifically
designed for separating and concentrating genomic
DNA and PCR products by ultrafiltration.
Retention
Retention, also sometimes called rejection, is a
function of molecular size and shape. Nominal cut-
off levels, defined with model solutes, are conven-
ient indicators. Degree of hydration, counter ions,
and steric effects can cause molecules with similar
molecular weights to exhibit very different retention
behavior. Many biological macromolecules tend to
aggregate, or change conformation under varying
conditions of pH and ionic strength, so that effective
size may be much larger than the “native” molecule,
causing increased rejection. Solute/solvent and
solute/solute interactions in the sample can also
change effective molecular size. For example, some
proteins will polymerize under certain concentration
and buffer conditions while others (e.g., heme
proteins) may break into corresponding subunits.
Ionic interactions or π – π stacking can cause small
molecules to behave similarly to molecules of
greater molecular weight. When this occurs, as in
the case of phosphate ions with a 500 NMWL
membrane, the small molecules may not effectively
permeate the membrane.
Millipore recommends the selection of a
membrane filter NMWL that is one half the size
of the molecule of interest. Other manufacturers
may recommend a smaller differential between the
size of the NMWL and the size of the molecule
but Millipore’s recommendation is designed to
provide maximum recovery. Please see additional
information regarding membrane NMWL selection
on page 4.
Concentration Polarization
Another factor affecting the retention characteristics
is the potential for membrane fouling, or concentra-
tion polarization. This occurs when there is an
accumulation of the retained solute on the surface
of the membrane. At high concentrations, a gel
layer forms that can act as a secondary membrane
(Figure 1.4). This may interfere with passage of the
molecules through the membrane and can adversely
affect the flow rate. In addition, pH, buffer compo-
nents, and concentration can result in a protein
behaving in an anomalous manner in terms of its
retention or passage by UF membranes.
During concentration polarization, the gel layer
on the membrane surface superimposes its own
rejection characteristics on those of the membrane.
Usually, concentration polarization increases reten-
tion of lower-molecular weight species. A membrane
with a 100K NMWL may reject 10 –20% of
albumin in a 0.1% solution of pure albumin.
However, in the presence of larger solutes such as
IgG, it may reject 90% of the albumin. Concentration
polarization makes it very difficult to use UF for
solute fractionation unless the solutes to be separated
differ in size by at least an order of magnitude.
Figure 1.4 Ultrafiltration separates proteins
from soluble salts. “Concentration polarization”
slows down filtration. The proteins form a gel layer
on the membrane surface.
5
 Flux (UF Flow Rate)
During ultrafiltration, it is important to balance speed
with retention to obtain optimal performance. A
membrane’s flux is defined as the flow rate divided
by the membrane area. Using membranes with
higher NMWL ratings will increase the flow, but at
the same time lower the retention. A membrane
should be selected for required rejection, consistent
with desired flow rate. This is determined by surface
area, macrosolute type, solubility, concentration and
diffusivity, membrane type, temperature effects on
viscosity and, to some extent, pressure. When
concentration polarization is rate-controlling, flux is
affected by solute concentration, fluid velocity, flow
channel dimensions, and temperature.
Effects of Operating Parameters on Flux
Pressure
When ultrafiltering dilute protein solutions or colloid
suspensions, flux will increase with increasing
transmembrane pressure (TMP). These effects are
most apparent when operating under controlled
positive pressure, such as when using a stirred cell.
When the process is membrane-controlled (i.e.,
when the resistance of the gel layer is much smaller
than that of the membrane), the flux-pressure
relationship is linear. When the process is controlled
by polarization (e.g., when the resistance of the
gel layer is much larger than that of the membrane),
flux will reach a plateau and may actually decrease
with increases in pressure.
Concentration
When concentration of the retained species is very
low, flux is independent of concentration. As solute
concentration rises during operation, increased
viscosity and the polarization effect cause flux to
decrease.
Temperature
Increasing the operating temperature normally
increases UF rates. A higher temperature increases
solute diffusivity (typically 3 –3.5% per degree
Celsius for proteins) and decreases solution
viscosity. Common practice is to operate at the
highest temperature tolerated by the solutes and
the equipment.
An exception to the rule is fermentation broth
concentration in the presence of some antifoams.
6
Many antifoams exhibit a phenomenon called
“cloud point.” As temperatures increase, antifoam
comes out of solution, forming a second phase.
Increasing temperature above the cloud point
causes flux to decrease.
pH
Changing solution pH often changes molecular
structure. This is especially true for proteins. At its
isoelectric point, a protein begins to precipitate,
causing a flux decrease.
Fouling
Flux decrease due to concentration polarization
should not be confused with the effect of membrane
fouling. Fouling is usually the deposition and
accumulation of submicron particles and solute on
the membrane surface and/or crystallization and
precipitation of smaller solutes on or within the pores
of the membrane. There may be a chemical
interaction with the membrane.
Importance of Recovery
While rejection is used to characterize membrane
performance, it does not always directly correlate
with solute recovery from a sample or volume.
Actual solute recovery—the amount of material
recovered after ultrafiltration—is generally based
on mass balance calculations.
In many cases, especially when working with
small samples of dilute, valuable solutions, the
degree of recovery of a target solute is vitally
important. In such cases, potential loss by non-
specific adsorption must be considered.
Different membrane materials adsorb biomole-
cules to varying degrees. Where maximum recovery
is desired, the choice of a membrane with the least
non-specific adsorptivity is essential. Millipore’s
Ultracel® regenerated cellulose membranes were
specifically developed to minimize non-specific
adsorption.
Since adsorption is a direct function of mem-
brane and device surface area, device size must be
considered when recovery is important. Small, dilute
samples should be concentrated with membranes of
minimal surface area, commensurate with achieve-
ment of reasonable flow rates. Millipore offers a
wide range of centrifugal devices, stirred cells, and
tangential flow systems with an extensive choice of
membrane areas and NMWLs.
Mode of Operation
The pressure required for ultrafiltration can be
supplied in a number of different ways depending
on the product in use. For example, Millipore's
small volume ultrafiltration products generally use
centrifugal force. Pump pressure is used with the
tangential-flow filtration (TFF) products and com-
pressed gas is utilized with the stirred cell products.
In addition, Millipore provides multiwell ultrafiltration
products that utilize vacuum and centrifugation.
Normal vs. Tangential Flow Filtration
Filtration can be broken down into two different
operational modes: normal flow filtration (NFF)
and tangential flow filtration (TFF). The difference
in fluid flow between these two modes is shown
in Figure 1.5.
Diafiltration
Millipore membranes provide an inexpensive means
of separating macromolecular mixtures into size-
graded classes either by direct ultrafiltration or by
diafiltration. Diafiltration removes microsolutes by
adding solvent to the solution being ultrafiltered
at a rate equal to the UF rate, independent of
microspecies concentration. This rapid, efficient
process washes microspecies from the solution at
constant volume, thereby purifying the retained
species. This process is most effective if the passing
Figure 1.5 Normal flow filtration (NFF) vs. tangential flow filtration (TFF)
In normal flow filtration (NFF), fluid is convected directly toward the
membrane under an applied pressure. Particulates that are too large
to pass through the pores of the membrane accumulate at the
membrane surface or in the depth of the filtration media, while smaller
molecules pass through to the downstream side. This type of process is
Normal Flow Filtration Tangential Flow Filtration
Feed Flow Pressure Pressure
Feed Flow
Membrane Membrane
Filtrate Filtrate
molecules are at least 10 times smaller than the
molecules to be retained and concentrated by the
membrane. Diafiltration is useful for sample
desalting and buffer exchange.
When diafiltration is used for sample desalting
or buffer exchange, there is no resulting change in
buffer composition. A solution volume with 100 mM
salt still contains 100 mM salt after the initial
concentration spin. Rediluting the retentate with
water and spinning again effectively decreases
the salt concentration of the sample by the con-
centration factor of the ultrafiltration. For example,
if a 4,000 µL sample containing 100 mM salt is
concentrated to 50 µL (80X) in an Amicon® Ultra
centrifugal filter unit, rediluted with water to
4,000 µL, and reconcentrated, the salt concen-
tration will be reduced 80X to 1.25 mM.
To achieve more complete salt removal, multiple
concentration and redilution spins are required.
For most samples, two concentration/reconstitution
cycles will remove about 99% of the initial salt
content.
With very small sample volumes, dilution of the
sample before the initial concentration spin can
often decrease salt concentration to an acceptable
level. For example, if a 200 µL sample containing
100 mM salt is diluted to 4,000 µL before concen-
tration in an Amicon Ultra centrifugal filter unit,
the salt concentration in the 4,000 µL sample will
be 5 mM. The concentrate will still contain 5 mM
often called dead-end filtration. However, the term “normal” indicates
that the fluid flow occurs in the direction normal to the membrane
surface, so NFF is a more descriptive and preferred name. NFF can
be used for sterile filtration of clean streams, clarifying prefiltration,
and virus/protein separations.
In tangential flow filtration (TFF), the fluid is pumped tangentially
along the surface of the membrane. An applied pressure serves to
force a portion of the fluid through the membrane to the filtrate side.
As in NFF, particulates and macromolecules that are too large to pass
through the membrane pores are retained on the upstream side.
However, in this case the retained components do not build up at the
surface of the membrane. Instead, they are swept along by the
tangential flow. This feature of TFF makes it an ideal process for finer
sized-based separations. Although TFF is more commonly associated
with large scale processing, centrifugal UF devices with vertical
membrane panels, such as Amicon Ultra devices, also benefit from a
TFF-like mode of separation, particularly in a swinging bucket rotor.
TFF is also commonly called cross-flow filtration. However, the term
“tangential” is descriptive of the direction of fluid flow relative to the
membrane, so it is the preferred name.
7
 salt. If more complete salt removal is desired, a
re-dilution/spin cycle should be added. In this
example, if the original spin ended with 50 µL of
retentate, redilution to 4,000 µL results in 0.06 mM
salt concentration. The sample can then be recon-
centrated to 50 µL in an Amicon Ultra centrifugal
filter device.
Diafiltration can be a continuous or a discon-
tinuous process. In continuous diafiltration, such
as in a stirred cell or a TFF device, the solution is
maintained at a fixed volume while solvent flows
continuously through the mixture. Salts and other
microsolutes are steadily removed by convective
transport. Microsolute exchange can be accom-
plished using the same principle. Constant operator
attention is not required and the possibility of solute
denaturation by overconcentration is eliminated.
In discontinuous diafiltration, such as in a centrifugal
ultrafiltration device, salts and microsolutes are
removed by repeated concentration and dilution
(Figure 1.6).
100 µL
10 µL
UF membrane
10X
concentration 90 µL
of protein
100 mM 100 mM
NaCl NaCl
Figure 1.6 Removing salts from retained solutes using diafiltration. The sodium chloride concentration is
reduced by dilution.
Table 1.1 Comparision of diafiltration and dialysis
Diafiltration
Transport convective with solvent,
independent of microsolute composition.
Rapid rate. Fractional removal
independent of content.
Ultrafiltration rate reduced with decreased
temperature (net effect not as marked).
At elevated macrosolute content,
ultrafiltration rate reduced.
Minimal exchange solvent required;
easily contained in reservoir.
Simple automation with endpoint
control apparatus.
8
Dialysis vs. Diafiltration
Dialysis is a traditional method for removing
microsolutes or exchanging solvents. It is a slow
diffusive process generally employing regenerated-
cellulose tubing as the barrier membrane. In dialysis,
the process solution and exchange solvent are on
opposite sides of the semi-permeable barrier
membrane through which permeating microsolutes
diffuse. The permeation rate of solutes from sample
to dialysate is a direct ratio to the solute concen-
tration and inversely proportional to the solutes’
molecular weight. Desalting by dialysis is time-
consuming and relatively inefficient at low
concentrations.
Millipore’s Amicon centrifugal concentrators
provide a fast, convenient, high-recovery alternative
to dialysis or precipitation without diluting samples.
The relative merits of diafiltration and dialysis are
summarized in the Table 1.1.
Add 90 µL 100 µL
10 µL
Milli-Q® water
10-fold 10X
dilution concentration 90 µL
of protein
10 mM 10 mM
NaCl NaCl
Dialysis
Transport diffusion-controlled,
dependent on type of microsolute.
Slow transport. Lower efficiency with
decreased microsolute concentration.
Marked temperature dependence
(reduced transport at lower temperature).
Microsolute transport relatively unaffected
by macrosolute content.
Frequent dialysate change. Recirculation
about bags to maximize transport.
Automation possible with complex equipment.
Guide to Selecting Membranes and Devices
Millipore offers a complete range of centrifugal and particulate materials. Durapore membranes are
devices used for sample concentration, purification, extremely hydrophilic, and they provide the lowest
and desalting or buffer exchange of soluble macro- binding of proteins and other biologicals of all
molecules. Millipore products are also available for commercially available microporous membranes.
applications such as cleaning up PCR reactions,
separating protein-bound from free ligands, Membranes Organized by
removing restriction enzymes, and recovering Typical Recoveries
oligonucleotides from agarose gels. This section For globular proteins, there is a good correlation
provides information to aid in choosing the correct between molecular weight and Stoke’s radius.
product for a particular application. This usually allows one to predict the recovery
of a protein based on its molecular weight if a
Membrane Selection membrane with the same nominal molecular weight
Millipore offers three distinct types of membranes to rating is used (see typical recovery of a panel of
choose from. This section will describe these three protein solutes in Table 2.1). However, in order to
types of membranes and then provide information accommodate the wide range of potential protein
about choosing the correct membrane based on solutes with different tertiary structures, we suggest
typical recoveries or application. initially using the “rule of two” to ensure optimal
recovery.
Types of Membranes
Rule of Two
Ultracel Ultrafiltration Membrane
For Ultracel (regenerated cellulosic) membranes,
To concentrate or desalt dilute solutions, use Millipore recommends using a membrane with
Ultracel series regenerated cellulose ultrafiltration a NMWL at least two times smaller than the
membranes. The hydrophilic, tight microstructure molecular weight of the protein solute that one
of Ultracel membranes assures the highest possible intends to concentrate.
retention with the lowest possible adsorption of
protein, DNA or other macromolecules. Rule of Three
For Biomax (polyethersulfone) membranes for
Biomax® Ultrafiltration Membrane
stirred cells and TFF, Millipore recommends using
To concentrate or desalt higher volumes of more
concentrated samples (recommended for protein
concentrations greater than 1.0 mg/mL), use Table 2.1 Membrane selection by recovery
Biomax polyethersulfone (PES) ultrafiltration mem- Cytochrome c BSA IgG
branes. Biomax membranes are recommended for NMWL (12.4 kDa) (67 kDa) (156 kDa)
samples such as serum, plasma, or conditioned 3K Q Q Q
tissue culture media.
10K P Q Q
Durapore® Microporous Membrane 30K • Q Q
To clarify biological samples, recover DNA from 50K • P Q
agarose gels, retain chromatography resins or 100K • • P
suspended solid media, use Durapore hydrophilic
PVDF microporous membranes. Durapore mem- Q Recommended (>90% recovery)
P Typically >90% recovery, depends on solute
branes allow all soluble protein and nucleic acids to
• Not recommended
pass, retaining sub-cellular fragments, whole cell

9
 a membrane with a NMWL at least three times
smaller than the molecular weight of the protein
solute that one intends to concentrate.
Membranes Organized by Application
Device Selection
See Table 2.3 to determine which centrifugal
product to use for protein concentration based on
initial sample molecular weight and volume.

See Table 2.2 to determine which centrifugal

product to use based on application and membrane.

Table 2.2 Membrane selection by application Ultrafiltration Membranes Microporous Specialty

Membranes Devices
Type* Molecular Weight
(NMWL) Pore Size (µm)

Montage Gel Extraction

Montage PCR
Micropure-EZ
Ultracel
Biomax

100K

0.45
0.65
10K
30K
50K

0.1
0.2

5.0
3K
5K
Protein concentration • • • • • • • •
Protein purification/desalting/buffer exchange • • • • • • •
Desalting of column fractions • • • • • • •
Protein isolation from cell lysates • • • •
Peptide concentration/desalting/buffer exchange • • •
Antibody concentration • • • •
Virus concentration or removal • • • •
Nucleic acid concentration/desalting/buffer exchange • • • • • • •
Oligonucleotide concentration/desalting/buffer exchange • •
PCR cleanup • • • • •
Remove linkers prior to cloning • • •
Remove labeled nucleotides • • • •
Antibody purification from hybridoma cells • • • •
Rapid restriction mapping • •
Clarify samples of particulate prior to HPLC • •
Clarification of cell lysates and tissue homogenates • • • • •
Cell harvesting • • • • •
Natural product screening • • • • • • • •
Restriction enzyme removal •
Bound vs. free drugs from serum/plasma (protein removal) • • •
DNA/RNA recovery from polyacrylamide gel • •
DNA recovery from agarose gel •
Oligonucleotide recovery from polyacrylamide gel • •
Removal of unincorporated label (e.g., fluorescein)
from protein • • • • • • •
Removal of imidazole from His-tag fusion protein • • • • • • •
*Selection of ultrafiltration membrane: Ultracel regenerated cellulose membrane is ideal for protein samples and nucleic acids.
Biomax polysulfone membrane is ideal for complex samples (e.g., serum).

10
Table 2.3 Protein concentration devices by filtration capacity

Filtration Capacity
Millipore Device 0.5 mL 2 mL 4 mL 15 mL 20 mL 70 mL 1L 2L 10 L ≥ 10 L

Small Volume Filtration Devices

Microcon® Centrifugal Filters*

Ultrafree ® 0.5 Centrifugal Filters
Ultrafree-MC Centrifugal Filters
MultiScreen® Filter Plate with Ultracel Membrane**
Medium Volume Filtration Devices

Centricon ® Centrifugal Filters

Ultrafree-CL Centrifugal Filters
Amicon Ultra-4 Centrifugal Filters
Amicon Ultra-15 Centrifugal Filters
Centriprep® Centrifugal Filters
Centriplus ® Centrifugal Filters
Centricon Plus-20 Centrifugal Filters
Large Volume Filtration Devices

Centricon Plus-70 Centrifugal Filters

Amicon Stirred Cells

Series 8000, High Output, Solvent-Resistant Stirred Cells

Tangential Flow Filtration (TFF)

Pellicon® XL 50 Ultrafiltration Devices

Prep/Scale® Spiral Wound UF Modules
Pellicon-2 Ultrafiltration Modules
*Bold type indicates recommended devices.
**96-well plate. Volume per well.

11
Small Volume Devices
Ultrafiltration Devices
Microcon Centrifugal Filters
Maximum initial volume: 500 µL
Typical final volume: 5 µL
NMWLs: 3K, 10K, 30K, 50K, 100K
Microcon centrifugal filter devices, the lab
standard, simply and efficiently concentrate
and desalt macromolecular solutions using
any centrifuge that can accept 1.5 mL tubes. The device’s low-
adsorption components and its Ultracel-YM membrane, together
with the patented invert recovery spin, combine to yield unusually
high recovery rates—typically > 95%, with concentration factors
as high as 100X. The invert spin also permits the recovery of
low volumes.
Ultrafree-0.5 Centrifugal Filters
Maximum initial volume: 500 µL
Typical final volume: 50 µL
NMWLs: 5K, 10K, 30K, 50K, 100K
Ultrafree-0.5 centrifugal filter devices
process aqueous biological solutions
using any centrifuge that accommodates
2.2 mL centrifuge tubes. The device’s vertical Biomax PES mem-
brane configuration, parallel to the direction of the centrifugal
force, reduces concentration polarization. This allows for the
highest possible flow rates, even in solutions with high levels of
particles. Concentrated protein is retrieved by pipette from a
concentrate “pocket” located below the membrane surface.
12
MultiScreen Filter Plates with
Ultracel-10 Membrane
Maximum initial volume: 500 µL per well
NMWL: 10K
MultiScreen Filter Plates with Ultracel-10
Membrane are the first automation-
compatible, high throughput ultrafiltration
plates for protein purification. The 10K
NMWL Ultracel regenerated cellulose ultrafiltration membrane
provides low non-specific binding and high protein recovery
(>95% retention of Cytochrome c).
Microfiltration Devices
Ultrafree-MC Centrifugal Filters
Maximum initial volume: 400 µL
Typical final volume: 5 µL
Pore sizes: 0.1 µm, 0.22 µm, 0.45 µm,
0.65 µm, 5.0 µm
Ultrafree-MC centrifugal filter units are
disposable devices used for sample
clarification with high recovery of biological solutions in the
0.05 to 0.5 mL range. Ultrafree-MC units are available in a
range of microporous and ultrafiltration membranes and are for
single use only. They fit standard microcentrifuge, fixed-angle
rotors capable of holding 1.5 mL or 20 mm outer diameter (O.D.)
tubes. Ultrafree-MC centrifugal filter units provide fast filtration and
highly reproducible performance.
Medium Volume Devices
Ultrafiltration Devices
Amicon Ultra-4 Centrifugal Filters*
Maximum initial volume: 4 mL
Typical final volume: 50 µL
NMWLs: 5K, 10K, 30K, 50K, 100K
Amicon Ultra centrifugal filters are the premier
tool for protein concentration, desalting, and
removing macromolecules and precipitates.
The devices combine Ultracel low-binding
ultrafiltration membrane with a vertical housing for fast sample
processing and typical sample recoveries of >90%. For volumes
up to 4 mL, the Amicon Ultra-4 device provides fast ultrafiltration
with the capability for high concentration factors.
Centricon Centrifugal Filters
Maximum initial volume: 2 mL
Typical final volume: 25 µL
NMWLs: 3K, 10K, 30K, 50K, 100K
Centricon centrifugal filter devices provide
fast, efficient concentration and desalting of
macromolecular solutions by ultrafiltration
through low-adsorption, hydrophilic
Ultracel-YM regenerated cellulose membranes. Designed for use
in centrifuges with fixed-angle rotors, they can provide up to
80-fold sample enrichment with minimal solute loss by adsorption.
Centriprep Centrifugal Filters
Maximum initial volume: 15 mL
Typical final volume: 700 µL
NMWLs: 3K, 10K, 30K, 50K
Centriprep centrifugal filter devices are
disposable ultrafiltration devices used for
concentrating and desalting high solute
biological samples. These complete,
ready-to-use ultrafiltration devices are designed for operation in
most centrifuges that can accommodate 50 mL centrifuge tubes.
Centriprep devices are best suited for applications in which
concentration factors are so high that they might affect protein
precipitation or aggregation.
Centriplus Centrifugal Filters
Maximum initial volume: 15 mL
Typical final volume: 500 µL
NMWLs: 3K, 10K, 30K, 50K, 100K
The disposable Centriplus centrifugal filter
device is used for the concentration and
desalting of macromolecular solutions with up
to 100-fold sample enrichment with minimal
loss of solute. These devices are designed for use in most centrifuges
that can accommodate 50 mL centrifuge tubes, with swinging-
bucket or fixed-angle rotors.
Amicon Ultra-15 Centrifugal Filters*
Maximum initial volume: up to 15 mL
Typical final volume: 200 µL
NMWLs: 5K, 10K, 30K, 50K, 100K
Amicon Ultra-15 centrifugal filters contain a
high recovery Ultracel membrane and are
used to concentrate biological samples, to
purify macromolecular components found in tissue culture extracts
or cell lysates, and for desalting and buffer exchange. The devices
can concentrate and desalt 15 mL in as few as 10 minutes.
Centricon Plus-20 Centrifugal Filters
Maximum initial volume: 20 mL
Typical final volume: 200 µL
NMWLs: 5K, 10K, 30K, 100K
Centricon Plus-20 centrifugal filter units are
disposable, single-use devices designed for
rapid processing of aqueous biological
solutions. The devices can concentrate most 20 mL solutions
down to a minimum volume of 200 µL.
Microfiltration Devices
Ultrafree-CL Centrifugal Filters
Maximum initial volume: 2 mL
Typical final volume: 10 µL
Pore sizes: 0.1 µm, 0.22 µm, 0.45 µm,
0.65 µm, 5.0 µm
Ultrafree-CL centrifugal filter units are
disposable, single-use devices used to process aqueous biological
solutions. Ultrafree-CL units can be used in standard, variable-
speed centrifuges with fixed-angle rotors capable of holding
15 mL tubes. A complete device consists of a filter cup and a
filtrate collection tube with cap. Ultrafree-CL units are available in
a range of microporous and ultrafiltration membranes.
*Amicon Ultra-4 and 15 are the first centrifugal devices to combine
high speed and high recovery. They are highly recommended for
most protein concentration and desalting applications.
13
Large Volume and TFF Devices
Ultrafiltration Devices
Centricon Plus-70 Centrifugal
Filters
Maximum initial volume: 70 mL
Typical final volume: 350 µL
NMWLs: 5K, 10K, 30K, 100K
Centricon Plus-70 centrifugal filter units
concentrate most 70 mL solutions down to
350 µL in just 25 minutes, making them a convenient alternative
to dialysis, lyophilization, precipitation techniques, or stirred cells.
Samples are typically concentrated in the 50X to 200X range,
depending on the sample type and starting sample volume.
Ta n g e n t i a l F l o w F i l t r a t i o n ( T F F )
Pellicon XL Devices
Maximum initial volume: 2 L
NMWLs: 5K, 8K, 10K, 30K, 50K,
100K, 300K, 500K, 1000K
Pellicon XL devices are small volume
tangential flow filtration (TFF) devices
offering performance, ease of use, and
linear scalability for pharmaceutical and
biotechnology development applications.
Pellicon XL devices are available in a wide selection of membrane
types and cut-offs. An optional graduated 100 mL reservoir is
available for simple small-volume separations.
Prep/Scale Spiral Wound
Ultrafiltration Modules
Maximum initial volume: 2 L to 10 L
NMWLs: 1K, 3K, 5K, 10K, 30K, 50K,
100K, 300K
Prep/Scale modules are used in conjunc-
tion with a peristaltic pump, flexible tubing
and feed solution in order to economically
prepare laboratory scale samples up to small production volumes
by tangential flow filtration. Highly reliable and consistent,
Prep/Scale modules offer cassette-like efficiencies with
reproducible separations performance.
Pellicon 2 Modules
Volumes >10 L
NMWLs: 1K, 3K, 5K, 8K, 10K, 30K,
50K, 100K, 300K, 500K, 1000K
Pellicon 2 modules offer true linear
scalability from laboratory size cartridges
to industrial cartridge assemblies for processing thousands of liters.
Pellicon 2 modules facilitate predicatable and faster scale-up with
less effort and are available with membranes to match virtually
any separation challenge.

Stirred Cells
Maximum initial volume: 2,000 mL Series 8000 Stirred Cells
Typical final volume: 3 mL • Five different sizes handle volumes from 3 mL to 400 mL
NMWLs: 1K, 3K, 10K, 30K, 50K, 100K, 300K, 500K • High flow rates with solutions up to 10% macrosolute
Stirred Cells are ideal for concentration, diafiltration and buffer concentration
exchange of macromolecule solutions including proteins, enzymes,
antibodies and viruses. They are capable of rapid concentration High-Output Stirred Cells
or salt removal followed by concentration in the same unit. • For 150 mm disc filters
Millipore offers three types of stirred cells: • Large membrane area

Solvent-Resistant Stirred Cells

• For 47 and 76 mm disc filters
• Borosilicate glass cylinder and PTFE components
for broad compatibility

Series 8000, High-Output and Solvent-Resistant Stirred Cells

14
Specialty Devices and Kits
Montage DNA Gel Extraction Kit
and Ultrafree-DA Centrifugal Filters
Designed to extract DNA fragments that are
100 to 10,000 bp in size; 0.45 µm Durapore membrane
The Montage DNA Gel Extraction Kit is a fast, effective solution
for fully functional DNA recovery from agarose gel slices. In one
10-minute spin, the
agarose gel material
containing the DNA
of interest is frag-
mented and com-
pressed to extrude
the DNA that is ready
for sequencing or
cloning. The Montage Gel Extraction kit contains all the necessary
consumables and the kit protocol is optimized for use with
standard agarose gels (<1.25% concentration). Ultrafree-DA
centrifugal devices available separately.
Montage PCR Centrifugal Filters
Maximum initial volume: 500 µL
Typical final volume: 100 µL
NMWLs: 3K, 10K, 30K, 50K, 100K
The Montage PCR filter unit is a convenient
method for single-sample PCR purification.
The high-performance device purifies PCR
products from salts and primers in a single
centrifugation step. Purified samples are
ready for downstream applications with no
additional purification steps.
Micropure ® -EZ Centrifugal Filters
Maximum initial volume: 250 µL
Holdup volume: < 5 µL
NMWLs: 3K, 10K, 30K, 50K, 100K
Micropure-EZ centrifugal filters provide an
easy, rapid means of removing restriction
and other enzymes from double-stranded
(ds) DNA. In a single 30-second spin, enzymes from the reaction
mix are selectively adsorbed by the membrane and DNA is
recovered in a vial—undiluted and enzyme-free. If concentration
or desalting of the sample is required, a Microcon centrifugal
filter device may be substituted for the filtrate vial, providing for
DNA purification, concentration and desalting in a single unit.
DNA recovery is not affected by the binding capacity of an
adsorption matrix. Micropure-EZ devices will deliver a minimum
of 85% recovery of dsDNA; higher recoveries can be achieved
by adding a rinsing step.
Centrilutor Micro-Electroeluters
Maximum initial volume: 2 mL
Typical final volume: 25 µL
NMWLs: 3K, 10K, 30K, 50K, 100K
The Centrilutor Micro-Electroeluter
elutes proteins from gel slices and then
removes the buffer and concentrates the
sample using Centricon centrifugal filter devices. This fast, easy
way to recover 1– 25 µg of protein from gel slices requires
no additional handling of the sample once the gel slices are
placed into the eluter.
15
Protocols • Proteins

Concentration, Desalting, and Buffer Exchange

with Amicon Ultra or Microcon Centrifugal Filters

Introduction Two examples below demonstrate the use of

Amicon centrifugal devices from Millipore are
ideal for removal or exchange of salts, sugars,
nucleotides, and non-aqueous solvents, as well as
other materials of low molecular weight. They also
serve to separate free from bound species.
Millipore centrifugal concentrators provide fast,
convenient, high-recovery alternatives to dialysis
and ethanol precipitation. Sample dilution, often
associated with spin columns, is not a problem.
Salt transfer across the membrane is efficient and
independent of microsolute concentration or size.
Millipore’s Amicon Ultra-4 and -15 centrifugal
filters are designed for high speed with high
recovery. The devices incorporate low-binding
Ultracel regenerated cellulose ultrafiltration mem-
brane for sample concentration and purification of
solutions containing dilute or purified protein solutes,
antigens, antibodies, enzymes, or microorganisms.
Their speed and excellent recovery
make them ideal for desalting and buffer exchange
applications. One of the most common applications
for Amicon Ultra devices is concentration and
desalting of column fractions during protein
purification by various chromatography methods.
Amicon Ultra devices for high protein and
enzymatic activity recovery.
Method
1. Select the device with the appropriate
NMWL and volume for the application.
2. Add the sample to the reservoir of the centrifugal
device.
3. If the sample is smaller than the maximum
volume, it can be diluted up to the maximum
volume before the first centrifugation step.
This will help increase the salt removal.
4. Centrifuge at the specified g-force for the
recommended amount of time.
5. Remove the initial filtrate from the filtrate tube
and set aside.
6. Add enough buffer or water to the device to
bring the sample volume up to 4 or 15 mL.
7. Centrifuge again.
8. Set aside the filtrate.
9. Recover the concentrated, de-salted sample.
NOTE: Both of the filtrates should be retained until
the concentrated sample has been analyzed.

Table 3.1 Removal of sodium chloride and recovery of protein with Amicon Ultra-15 device
Cytochrome c Cytochrome c BSA BSA IgG
0.25 mg/mL 0.25 mg/mL 1 mg /mL 1 mg/mL 1 mg/mL
NMWL 5 kDa 10 kDa 30 kDa 50 kDa 100 kDa
Spin % Protein % NaCl % Protein % NaCl % Protein % NaCl % Protein %NaCl % Protein % NaCl
Recovery Removal Recovery Removal Recovery Removal Recovery Removal Recovery Removal
1 94.5 97.2 97.3 97.9 96.0 98.2 98.9 97.1 99.9 97.7
2 92.6 99.9 95.6 99.9 94.4 99.9 92.4 99.9 97.1 99.5

Three Amicon Ultra-15 devices of each cut-off were tested with 15 mL of solute. 500 mM NaCl was added to each solution. Each spin was
performed at 4000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q® (Millipore)
system. OD readings were taken at 410 nm for Cytochrome c and 280 nm for BSA and IgG.

16
Results As the results show in Tables 3.1– 3.4 , the
efficient design of the Millipore devices allowed
The transfer of salts across a membrane filter is
> 90% of the salt to be removed during the first
independent of sample concentration or size. There
centrifugation step. Typically, only one subsequent
is no change in the composition of the buffer when
centrifugation step was needed to increase the
desalting using ultrafiltration. For example, a solution
typical salt removal to 99% with > 90% recovery of
containing 500 mM salt still contains that concen-
the sample.
tration after the initial centrifugation. Adding another
Protein purification by chromatography usually
volume of salt-free buffer or water to the retentate
involves the collection of multiple column fractions,
and centrifuging again will reduce the salt concen-
with only some of those fractions containing the
tration. This process, known as diafiltration, can be
protein of interest. After the fractions are combined,
repeated to achieve maximum salt removal.
a protein concentration step is often required for
Diafiltration can also be used when it is desirable to
protein storage, or concentration with buffer
have the sample in a different buffer. The sample is
exchange may be needed for downstream
concentrated and then repeatedly diluted with the
separations.
desired buffer and concentrated again.

Table 3.2 Removal of sodium chloride and recovery of protein with Amicon Ultra-4 device
Cytochrome c Cytochrome c BSA BSA IgG
0.25 mg/mL 0.25 mg/mL 1 mg /mL 1 mg/mL 1 mg/mL
NMWL 5 kDa 10 kDa 30 kDa 50 kDa 100 kDa
Spin % Protein % NaCl % Protein % NaCl % Protein % NaCl % Protein %NaCl % Protein % NaCl
Recovery Removal Recovery Removal Recovery Removal Recovery Removal Recovery Removal
1 100.0 95.2 97.0 93.0 96.4 96.4 98.5 92.1 100.0 99.2
2 95.8 97.9 95.6 99.5 93.7 99.7 99.3 99.7 99.4 99.7

Three Amicon Ultra-4 devices of each cut-off were tested with 4 mL of solute. 500 mM NaCl was added to each solution. Each spin was performed
at 4000 x g for 10 minutes. After the first spin, the retentate was brought up to 4 mL with ultrapure water from a Milli-Q (Millipore) system. OD
readings were taken at 480 nm for Cytochrome c and 280 nm for BSA and IgG.

Table 3.3 Removal of sodium chloride and recovery of IgG with Centricon Plus-20 filter device
with Ultracel-PL 30 membrane
Spin % NaCl % IgG
Number Remaining Recovered
1 1 —
2 0.4 89.8

19 mL of 1 mg/mL bovine IgG in 500 mM NaCl was concentrated to 150 µL. Sample was spun twice at
2000 x g in a swinging bucket rotor at 25 °C for 20 minutes.

Table 3.4 Removal of riboflavin and recovery of IgG with Microcon filter device with Ultracel-YM
membrane
Spin % Riboflavin % IgG
Number Remaining Recovered
1 9 95
2 2 93
3 <1 93
4 <1 92

500 µL of a 50:50 mixture of riboflavin and IgG were spun in a Microcon 3K NMWL device at 12,000 x g for 75
minutes at room temperature in 55° angle rotor. After the initial spin, the retentate was twice diluted with 500 µL of
PBS and spun again. After each spin, concentration of riboflavin and IgG in the filtrate and retentate were monitored.

17
 Concentration of Indoleamine
2,3-Dioxygenase
Courtesy of Eduardo Vottero,
University of British Columbia
Indoleamine 2,3-dioxygenase (IDO; MW 48,000)
is a heme-containing enzyme that is the first and
rate-limiting enzyme in human tryptophan metabo-
lism. IDO processes 98% of the total tryptophan
available in the human body and is critical in
suppression of immunoresponse by blocking
T-lymphocyte proliferation locally [Swanson et al,
Am J Respir Cell Mol Biol [manuscript in prepara-
tion] (2003); Sarkhosh et al, J Cell Biochem 90,
206 (2003); Mellor et al, J Immunol 171, (2003)].
Recombinant IDO was expressed in E. coli BL21
(DE3) cells utilizing the pET 28a (+) vector system.
In this system, a hexahistidyl tag was fused to full-
size IDO at the N-terminus with a spacer sequence
and a thrombin cleavage site. The protein was
purified by conventional His-tag purification methods
and eluted with imidazole. The histidine tag was
removed by thrombin cleavage. Final purification
was done by gel filtration chromatography G-75.
Amicon Ultra-15 centrifugal devices were used to
40 kDa
35 kDa
Figure 3.1 SDS-PAGE of purified indoleamine
2,3-dioxygenase before and after concentration
using Amicon Ultra-15 centrifugal devices.
Table 3.5 PKR concentration results
Spin 1
Starting volume (mL) 15
Starting concentration (mg/mL) 0.229
Volume of retentate (µL) 590
Total amount of PKR (mg)
Before UF 3.4350
After UF 3.1005
Filtrate 0.0000
% Recovery 90.26
18
concentrate the IDO fractions from an initial
concentration of ~ 0.5 mg/mL to a final concentra-
tion of 10 mg/mL. Samples were analyzed by
SDS-PAGE using a 12.5% polyacrylamide gel
(Figure 3.1). In addition, it was shown that no IDO
activity loss was observed after concentration
using an Amicon Ultra device.
Concentration of PKR
and Buffer Exchange
Courtesy of Peter A. Lemaire and
Dr. James Cole, University of Connecticut
Human protein kinase R (PKR) is one of the major
proteins induced by interferon as part of the host
defense against viral infection1–4. PKR is synthesized
in a latent form and is activated by autophosphory-
lation induced upon binding dsRNA. Once
phosphorylated, active PKR phosphorylates the
eukaryotic translation initiation factor elF2α leading
to a block in protein synthesis in virally infected
cells. PKR has been implicated as a participant in
various signal transduction pathways associated
with cellular processes including transcription7–9,
differentiation10, apoptosis11, splicing14 and
transformation5,6. However, difficulties in purifying
PKR in large amounts has limited rigorous biophys-
ical characterization of the mechanisms of PKR
activation. A high-yield prokaryotic expression
system has been developed for PKR, and PKR has
been purified using three chromatography steps on
Agarose-Heparin, Agarose-Poly (I), Poly (C) and
Sephacryl S-200 gel filtration columns. After the
last step, PKR-containing column fractions were
pooled and concentrated using Amicon Ultra-15
30K NMWL devices. The concentration step was
necessary for long-term protein storage. Table 3.5
shows the protein recovery results obtained after
four concentrations. Over 90% recovery was
obtained and no protein loss to the filtrate was
observed.
Spin 2 Spin 3 Spin 4
15 15 15
0.229 0.169 0.169
490 203 225
3.4350 2.5320 2.5320
3.1548 2.4081 2.3983
0.0000 0.0000 0.0000
91.84 95.11 94.72
 Amicon Ultra-15 30K NMWL devices were also
used for exchanging buffer for PKR autophosphory-
lation (activation) assay. 200 µL of 6.301 mg/mL
PKR in Protein Storage buffer (20 mM HEPES,
1 M NaCl, 10 mM β-mercaptoethanol, 0.1 mM
EDTA, 10% glycerol, pH 7.5) was diluted to 15 mL
with Phosphorylation Buffer (20 mM HEPES, 50 mM
KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT,
pH 7.5) and re-concentrated three times using
Amicon Ultra devices at 3000 x g for 20 minutes at
4 °C. The filtrates from the three steps were pooled
and the total amount of protein in all samples was
determined by UV absorption A280.
Protein activity was tested by autophosphory-
lation assay. The protein in the storage buffer was
supplemented with 5 mM MgCl2 and the samples
were allowed to undergo autophosphorylation at
30 °C for 20 minutes in the presence of 3 mM ATP
and 3 µCi [γ32P] ATP. The activity was determined
by autoradiography and quantified by liquid
scintillation counting. As shown in Figure 3.2, the
activity of PKR when no buffer exchange was only
6% of that when buffer exchange step was per-
formed. Hence, the UF successfully exchanged the
buffer while maintaining the activity of the protein.
References
1. Samuel CE. Antiviral actions of interferon.
Interferon-regulated cellular proteins and their
surprisingly selective antiviral activities.
Virology 1991;183(1):1–11.
2. Hovanessian AG. The double stranded RNA-
activated protein kinase induced by interferon:
dsRNA-PK. J Interferon Res 1989;9(6):641– 7.
100
Relative Activity of PKR
80
60
40
20
0
No
Buffer
Exchange
Figure 3.2 Comparison of PKR autophosphoryla-
tion with buffer exchange by ultrafiltration and
without buffer exchange.
3. Lebleu B, et al. Interferon, double-stranded RNA,
and protein phosphorylation. Proc Natl Acad Sci
USA 1976;73(9):3107–11.
4. Samuel CE. Mechanism of interferon action:
phosphorylation of protein synthesis initiation
factor eIF-2 in interferon-treated human cells by
a ribosome-associated kinase processing site
specificity similar to hemin-regulated rabbit
reticulocyte kinase. Proc Natl Acad Sci USA
1979;76(2):600–4.
5. Koromilas AE, et al. Malignant transformation by
a mutant of the IFN-inducible dsRNA-dependent
protein kinase. Science 1992;257:1685–9.
6. Meurs E, et al. Tumor supressor function of
interferon-induced double-stranded RNA
activated protein kinase. Proc Natl Acad Sci
USA 1993;90:232–6.
7. Wong AH, et al. Physical association between
STAT1 and the interferon-inducible protein kinase
PKR and implications for interferon and double-
stranded RNA signaling pathways. EMBO J,
1997;16(6):1291–304.
8. Cuddihy AR, et al. Double-stranded-RNA-
activated protein kinase PKR enhances tran-
scriptional activation by tumor suppressor p53.
Mol Cell Biol 1999;19(4):2475–84.
9. Demarchi F, Gutierrez MI, Giacca M. Human
immunodeficiency virus type 1 Tat protein
activates transcription factor NF-kappaB through
the cellular interferon-inducible, double-stranded
RNA-dependent protein kinase, PKR. J Virol
1999; 73(8):7080–6.
10. Petryshyn R, et al. Effect of interferon on protein
translation during growth stages of 3T3 cells.
Arch Biochem Biophys 1996;326(2):290 –7.
11. Barber GN. Host defense, viruses and apop-
tosis. Cell Death Differ 2001;8(2):113–26.
12. Tan SL, Katze MG. The emerging role
of the interferon-induced PKR protein kinase as
a apoptotic effector: A new face of death?
J Interferon Cytokine Res 1999;19:543–54.
13. Balachandran S, et al. Activation of the dsRNA-
dependent protein kinase, PKR, induces apop-
tosis through FADD-mediated death signaling.
EMBO J, 1998;17(23):6888–902.
14. Osman F, et al. A cis-acting element in the
3'-untranslated region of human TNF-alpha
mRNA renders splicing dependent on the
activation of protein kinase PKR. Genes Dev
Buffer 1999;13(24):3280–93.
Exchange
19
Protocols • Proteins

Detergent Removal with Microcon,

Amicon Ultra or Centricon Centrifugal Filters

Introduction Method and Results

Microcon, Amicon Ultra and Centricon centrifugal
filters are efficient laboratory tools for removing small
molecules from solutions of proteins or nucleic acids.
Often, the molecule to be removed is one of a
number of commonly used detergents or protein
solubilizing agents. The chemical nature of most
detergents allows for micelle formation above a
critical concentration limit (Critical Micelle
Concentration, CMC). Micelle formation results in
aggregation of the detergent and leads to gross
changes in molecular structure. This affects the
amount of the detergent that can be removed from a
solution by centrifugal devices with specific nominal
molecular weight limit (NMWL) membranes.
For example, the monomer of Triton® X-100
has a molecular weight of 500–650 daltons.
Triton X-100 should pass readily through the
10,000 NMWL membrane in an Amicon Ultra
device. However, at concentrations above
0.01% (0.2 mM), Triton X-100 forms micelles
composed of approximately 140 monomeric units.
During ultrafiltration, the micelles behave like
70,000–90,000 dalton globular proteins.
As a result, more than 90% of Triton is retained by
the ultra-filtration membrane. Therefore, above the
CMC of Triton X-100, an Amicon Ultra-4 100K
NMWL concentrator would be required to remove
the detergent effectively.
In a series of studies, Millipore researchers used
Total Organic Carbon (TOC) analysis to measure
detergent removal by Microcon, Amicon Ultra and
Centricon concentrators after a single centrifugation
spin (note that complete detergent removal generally
requires 3–5 spins). As the results in the Tables 4.1
and 4.2 indicate, detergent removal depends both
on the original detergent concentration and the
NMWL of the centrifugal units. All measurements
shown were made using detergent/distilled water
solutions.
NOTE: Temperature, the presence of salts in
the solution, and/or macromolecule/detergent
interactions may lower the CMC for a particular
detergent. Use the tables only as general
guidelines in assessing the efficiency of detergent
removal with the Microcon, Amicon Ultra and
Centricon devices.

20
Table 4.1 Percent detergent removal after one spin with Centricon and Table 4.2 Percent detergent removal
Microcon centrifugal devices after one spin with Amicon Ultra-4
centrifugal devices
NMWL NMWL
Detergent 3kDa 10 kDa 30 kDa 50 kDa 100 kDa Detergent 10 kDa 30 kDa
SDS SDS
0.01% > 90% > 90% > 90% > 90% > 90% 0.1% 95% 98%
0.1% > 90% > 90% > 90% > 90% > 90% 1% 38% 48%
1% 40 – 89% 40 – 89% 40 – 89% 40 – 89% 40 – 89% 5% 94% 95%
5% < 40% < 40% < 40% 40 – 89% 40 – 89% Tween-20
NaDeoxycholate 0.1% 30% 42%
0.1% > 90% > 90% > 90% > 90% > 90% 1% 28% 35%
1% > 90% > 90% > 90% > 90% > 90% 5% 82% 77%
5% 40 – 89% 40 – 89% 40 – 89% > 90% > 90% Triton X-100
CAPS 0.1% 3% 47%
5% > 90% > 90% > 90% > 90% > 90% 1% 2% 20%
CPCl1 5% 2% 20%
0.01% > 90% > 90% > 90% > 90% > 90% CHAPS
0.1% 40 – 89% 40 – 89% 40 – 89% 40 – 89% 40 – 89% 0.1% 90% 96%
1% < 40% < 40% < 40% < 40% 40 – 89% 1% 66% 93%
5% < 40% < 40% < 40% < 40% 40 – 89% 5% 38% 73%
TDMABr2
0.1% > 90% > 90% > 90% > 90% > 90%
1% < 40% < 40% < 40% 40 – 89% > 90%
5% < 40% < 40% < 40% < 40% > 90%
Digitonin
0.01% > 90% > 90% > 90% > 90% > 90%
0.1% 40 – 89% 40 – 89% 40 – 89% 40 – 89% 40 – 89%
1% < 40% < 40% < 40% < 40% < 40%
Tween®-20
0.01% < 40% < 40% 40 – 89% 40 – 89% > 90%
0.1% < 40% < 40% < 40% 40 – 89% > 90%
1% < 40% < 40% < 40% < 40% 40 – 89%
5% < 40% < 40% < 40% < 40% < 40%
Triton X-100
0.01% 40 – 89% 40 – 89% 40 – 89% 40 – 89% > 90%
0.10% < 40% < 40% < 40% < 40% > 90%
1% < 40% < 40% < 40% < 40% 40 – 89%
5% < 40% < 40% < 40% < 40% < 40%
CHAPS
0.10% > 90% > 90% > 90% > 90% > 90%
1% 40 – 89% 40 – 89% > 90% > 90% > 90%
5% < 40% < 40% 40 – 89% > 90% > 90%
1Cetylpyridinium chloride
2Tetradecyltrimethylammonium bromide

21
Protocols • Proteins

Two-Dimensional Electrophoresis Sample Preparation

with Amicon Ultra Centrifugal Filters
Two-dimensional electrophoresis (2DE) is one of the
most commonly used methods in proteome analysis.
Briefly, proteins are separated by their isoelectric
point (first dimension separation) followed by SDS-
PAGE separation by molecular weight (second
dimension separation). Although it is a powerful
method for simultaneously displaying hundreds of
proteins, 2DE presents a challenge for sample
preparation. Salts and ionic detergents are common
chemicals contaminating biological samples that
are not compatible with 2DE separation; however
they are often required to solubilize proteins from
cells and tissues. The high concentration of salts
combined with the relatively low protein content
make samples completely unsuitable for isoelectric
focusing.
Usually protein concentration is achieved by
protein precipitation with acetone or TCA. The
disadvantage of protein precipitation is that some
of the proteins become insoluble and can not be
resolubilized in IPG buffer. Another disadvantage is
that many salts become insoluble in acetone and
precipitate along with the proteins.
Ultrafiltration (UF) can achieve protein concen-
tration and desalting in one step. Figures 5.1 and
5.2 present two examples of protein preparation
for 2DE by acetone precipitation and ultrafiltration.
Both examples demonstrate that UF provides more
efficient salt removal and allows better separations
and improved resolution of protein spots in
two-dimensional electrophoresis.

Figure 5.1 Two-dimensional electrophoresis of yeast cell lysate prepared by acetone precipitation (left) and
ultrafiltration (right). Sacharomyces cerevisiae strain s288c was grown to log phase. The cells were pelleted
and resuspended in Cellular and Organelle Membrane Solubilizing Reagent from the ProteoPrep™ kit (Sigma)
and lysed by sonication. Cellular debris was removed by centrifugation and the supernatants were reduced
and alkylated with 5 mM tributylphosphine and 10 mM acrylamide. Lysates were acetone-precipitated to
remove residual Tris and alkylating reagent or were filtered through Amicon Ultra-4 10K NMWL devices.
Samples were redissolved in ProteomIQ™ Resuspension Reagent (Proteome Systems), focused in broad range
(pH 3 –10) immobilized pH gradients (IPGs) on the IsoelectrIQ™ IEF instrument. Second dimension gels were
6–15% polyacrylamide gradient GelChips™ (Proteome Systems) run on an ElectrophoretIQ™ 2D instrument
(Proteome Systems). Data courtesy of Dr. G. Smejkal, Proteome Systems, Inc., Woburn, MA, USA.

22
Figure 5.2 Two-dimensional electrophoresis of endothelial cell lysates prepared by acetone precipitation (left)
and ultrafiltration with Amicon Ultra devices (right). Whole cell lysates of endothelial cells were prepared in
7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 20 mM Tris buffer. The samples were very dilute and presum-
ably contained DNA fragments, salts and lipids. 300 mg of the total protein was either acetone-precipitated or
desalted in Amicon Ultra devices. After acetone precipitation with four volumes of cold acetone, samples were
incubated overnight at – 20 °C, precipitated by centrifugation, rinsed with cold acetone, and pelleted again.
The resulting pellet was resuspended in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT,
20 mM Tris). Alternatively, samples were filtered through Amicon Ultra-4 10K NMWL devices after being
mixed with nine volumes of 20 mM Tris HCl, pH 7. The sample was concentrated to approximately 40 µL
and adjusted to 350 µL with rehydration buffer. The proteins were separated by IEF in 18 cm IPG Drystrips™,
pH 3–10 (Amersham). A second dimension separation was performed in 10% SDS-PAGE gel. The gels
were silver stained and scanned. Data courtesy of Dr. Leonid Kryazhev, Genome Quebec, Montreal, Canada.

23
Protocols • Proteins
Purification of Serum Peptides for Biomarker Research
with Amicon Ultra Centrifugal Filters
Introduction
A biomarker can be defined as a molecule that
indicates an alteration in physiology. Biomarkers
play an essential role in the drug discovery and
development process. They provide powerful clues
to genetic susceptibility, disease progression, and
predisposition, as well as drug response and the
physiological and metabolic profiles of diseases
and drug responses. Biomarkers can also provide
valuable diagnostic and prognostic information that
can facilitate personalized medicine. Peptide and
protein patterns have been linked to ovarian cancer,
breast cancer, prostate cancer and astrocytoma1–5.
Most diagnostic tests are based on blood or
urine analysis5. Serum is a key source of putative
protein biomarkers, and, by its nature, can elucidate
organ-confined events. Use of mass spectrometry
coupled with bioinformatics has been demonstrated
as being capable of distinguishing between serum
protein pattern signatures in late-stage and early-
stage ovarian cancer patients6.
One of the major impediments to the discovery
of new biomarkers is the presence of salts, proteins,
and lipids in plasma or serum that makes it difficult
to detect and analyze peptides by mass spectrom-
etry. When untreated serum is spotted onto a
MALDI-TOF plate, it does not produce any useable
signal in mass spectrometry. Multiple protocols have
been developed to extract and enrich peptides from
tissues and body fluids, such as batch reversed phase
chromatography over C18 resin and extraction with
0.1% trifluoroacetic acid (TFA) or 50% acetonitrile to
selectively precipitate large proteins while enhancing
the solubility of smaller proteins and peptides.
Ultrafiltration has previously been reported as
a sample preparation tool to prepare low molecular
weight fractions for biomarker analysis7–10. In this
study we show that ultrafiltration in combination with
solid phase extraction (SPE) on C18 resin can be a
convenient and efficient method for serum peptide
purification. The approach provides more peptides
24
for mass spectrometry analysis than the acetonitrile
precipitation method.
Methods
Preparation of Serum Peptides by UF and SPE
One milliliter of human serum, with or without
acetonitrile, was filtered using Amicon Ultra-4
10K NMWL centrifugal devices. The devices were
centrifuged in a swinging bucket rotor for 15 to 30
minutes at 3000 x g. Ten microliters of the filtrate
was acidified with 5 µL of 1% TFA, concentrated
with ZipTip®µC18 pipette tips. Co-elution was
performed directly onto a MALDI target with 2 µL of
∂-cyano-4-hydroxycinnamic acid matrix (5 mg/mL
in 50% acetonitrile, 0.1% TFA). If acetonitrile was
added to the serum prior to the filtration, the samples
were briefly evaporated in a Speed-Vac™ centrifuge
to remove solvent before ZipTip purification.
Peptide Analysis by Mass Spectrometry
Peptide-containing ultrafiltrates from cell lysates or
human serum were acidified with 1% TFA and
concentrated on ZipTipµC18 or ZipTipSCX pipette
tips following the procedure outlined in the user
guide. All samples were overlaid with 1 µL of
∂-cyano-4-hydroxycinnamic acid matrix (5 mg/mL
in 50% acetonitrile, 0.1% TFA) and analyzed on
Voyager-DE™ Workstation (Applied Biosystems)
in linear mode.
Preparation of Serum Peptides
by Acetonitrile Precipitation
Acetonitrile was added to human serum at a
1:1 ratio (v:v), and samples were centrifuged to
precipitate larger proteins. The supernatant was
dried in a Speed-Vac centrifuge, resuspended in
0.1% TFA, and then desalted and concentrated
with ZipTipµC18 pipette tips. Co-elution was
performed directly onto a MALDI target with
2 µL of ∂-cyano-4-hydroxycinnamic acid matrix
(5 mg/mL in 50% acetonitrile, 0.1% TFA).
Results supernatant after 50% acetonitrile precipitation;
and (C) serum ultrafiltrate processed with an Amicon
While human serum contains numerous peptides
Ultra-4 30K NMWL device. We observed higher
and small proteins, they are not accessible by direct
quality spectra and an increased number of MALDI-
mass spectrometry analysis. Even after reverse
TOF detected peptides if the serum was ultrafiltered.
phase concentration and desalting, only a few
Further improvement of spectra can be
peptides are detectable in the mass spectrum
achieved by reverse phase chromatography,
(data not shown). This can be explained by the
which concentrates and desalts the peptides.
high concentration of proteins and lipids competing
The ZipTipµC18 pipette tip is a convenient and
with the peptides to bind to the resin (Figure 6.1A).
efficient tool for micro-scale sample preparation
One of the common methods for serum peptide
prior to mass spectrometry. Figure 6.2 shows the
preparation for mass spectrometry is acetonitrile
MALDI-TOF spectra of rat serum peptides prepared
fractionation, where the addition of 50 – 70%
with ZipTipµC18 pipette tip out of (A) straight serum,
acetonitrile precipitates larger proteins, while the
(B) 50% acetonitrile supernatant and (C) serum
peptides stay soluble in the supernatant. Another
processed by ultrafiltration. The last method provided
way to produce relatively protein-free filtrates is
stronger MALDI-TOF signal, higher signal-to-noise
ultrafiltration. Figure 6.1 presents the MALDI-TOF
ratio and double the number of detected peptides.
spectra of (A) straight rat serum; (B) serum

A B C

Figure 6.1 MALDI-TOF spectra of (A) unprocessed rat serum; (B) serum peptides in 50% acetonitrile supernatant; and (C) serum ultrafiltrate
processed with Amicon Ultra-4 30K NMWL centrifugal device.

Figure 6.2 MALDI-TOF spectra of rat serum

peptides after concentration and desalting by A B
reversed phase chromatography: (A) rat serum
processed with ZipTipµC18 pipette tip; (B) serum
supernatant after 50% acetonitrile precipitation
processed with ZipTipµC18 pipette tip; (C) 30K
serum ultrafiltrate processed with ZipTipµC18
pipette tip; and (D) the same as (C) but 20%
acetonitrile was added to the serum prior to
ultrafiltration.

C D

25
 We have also investigated whether the addition
of acetonitrile to serum prior to ultrafiltration improves
the detection of serum peptides (MALDI spectrum
shown in Figure 6.2D). Further improvement of the
spectrum was attained, especially in the m/z 1000
area of the spectrum.
Conclusion
For the analysis of serum peptides, complexity
reduction by eliminating higher molecular weight
proteins is critical for high resolution mass spec-
trometry. Efficient separation of peptides from the
majority of proteins and salts can be achieved by
sample ultrafiltration. We have shown the effective
use of Amicon Ultra-4 30K NMWL centrifugal
devices for the preparation of peptides. Other
molecular weight cut offs can be utilized depending
on the desired range of peptides. The method can
be used directly in combination with ZipTipµC18
pipette tips for peptide identification by MS/MS or
as a first step prior to further surface-mediated
enrichment using SELDI-TOF methods.
26
References
1. Ardekani AM, Liotta LA, Petricoin III. Expert
Rev Mol Diagn 2002;2:12.
2. Carter D, Douglass JF, Cornellison CD, Retter
MW, Johnson JC, Bennington AA, Fleming TP,
Reed SG, Houghton RL, Diamond TS, Vedvick
TS. Biochemistry 2002;41:6714.
3. Wellmann A, Wollscheid V, Lu H, Ma ZL,
Albers P, Schutze K, Rohde V, Behrens P,
Dreschers S, Ko Y, Wernert N. Int J Mol Med
2002;9:341.
4. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ,
Fusaro VA, Steiberg SM, Mills GB, Simone C,
Fishman DA, Kohn EC, Liotta LA. Lancet 2002;
359:572.
5. Bischoff R, Luider TM. J Chrom B 2004;803:
27–40.
6. Stevens EV, Liotta LA, Kohn EC. J Gynecol
Cancer 2003;13:133– 9.
7. Schulz-Knappe P, Schrader M, Standker L,
Richter R, Hess R, Jurgens M, Forssmann W-G.
J Chromatogr A 1997;776:125–132.
8. Basso D, Valerio A, Seraglia R, Mazzza S,
Piva MG, Greco E, Fogar P, Gall N,
Pedrazzoli S, Tiengo A, and Plebani M.
Pancreas 2002;24:8–14.
9. Prazeres S, Santos MA, Ferreira HG, Sobrinho
LG. Clin Endocrinol (Oxf) 2003;58:686–90.
10. Tirumalai RS, Chan KC, Prieto DA, Issaq HJ,
Conrads TP, Veenstra TD, Mol Cell Proteomics
2003;10:1096 –103.
 Protocols • Proteins

Rapid Antibody Concentration

with Amicon Ultra Centrifugal Filters
Ultrafiltration offers a fast and convenient way to
concentrate antibodies purified from serum, ascites
40 12
fluid, or hybridoma supernatants. The traditional

IgG Concentration (mg/mL)

purification protocol for immunoglobulins includes PROSEP-A
10
30 PROSEP-G
affinity binding to Protein A or G chromatography

IgG Volume (mL)

8
media, washing unbound proteins, and eluting with
a low pH buffer. Subsequently, purified antibodies 20 6

are often too dilute for their intended purpose or 4

for long-term storage. In addition, harsh elution 10
2
conditions sometimes require buffer exchange to
preserve protein activity. 0 0
0 3 10 15 20
Dialysis is often used for antibody concentration
Centrifugation Time (min)
and buffer exchange. However, ultrafiltration provides
a quick, alternative method to concentrate and
Figure 7.1 Concentration of rabbit IgG with Amicon Ultra-15 devices. The
diafilter immunoglobulins with up to 99% recovery IgGs were purified using Montage PROSEP-A or PROSEP-G Antibody Purification
and one-step salt removal (see Concentration, Kits. The lines show IgG volume reduction, while the bars show a proportional
Desalting, and Buffer Exchange with Amicon Ultra increase in IgG concentration after 20 minutes of centrifugation time.
and Microcon Centrifugal Filters, page 16).
To demonstrate the suitability of Amicon Ultra
devices for concentrating purified IgG, rabbit Figure 7.2 kDa

serum was processed with Montage PROSEP-A SDS-PAGE gel of purified rabbit 116
IgG before and after concentration
and PROSEP-G Antibody Purification Kits (Millipore)
with Amicon Ultra-15 devices.
and then concentrated with Amicon Ultra-15 30K 66
55
NMWL devices. Figure 7.1 shows a decrease in Lane 1: MW standards Heavy
Lanes 2, 3: PROSEP-A-purified IgG Chain
the retentate volume proportional to the increase in
before concentration 36
antibody concentration. A twenty-minute centrifuga-
(lane 2) and after
Light
tion resulted in > 95% recovery of immunoglobulins. concentration (lane 3)
21 Chain
Figure 7.2 shows an SDS-PAGE gel of purified Lanes 4, 5: PROSEP-G-purified IgG
14
rabbit IgGs before and after ultrafiltration. before concentration
(lane 4) and after
concentration (lane 5)
Protein Load: 5 µL in lanes 2 and 4;
1 µL in lanes 3 and 5 1 2 3 4 5

27
Protocols • Proteins
Removal of Unincorporated Label from
Labeled Protein with Amicon Ultra or
Centricon Centrifugal Filters
Introduction
Centrifugal devices containing ultrafiltration
membranes are ideal for the removal or exchange
of salts, sugars, nucleotides, non-aqueous solvents,
and other materials of low molecular weight.
They also serve to separate free from bound
species. One of the applications is removal of
unincorporated label in protein labeling applications
where Millipore centrifugal devices provide fast,
convenient, high-recovery alternatives to gel
filtration. Sample dilution, often associated with
gel filtration, is not a problem. A few rounds of
diafiltration can efficiently remove unincorporated
label. Two examples below demonstrate the use of
Millipore centrifugal ultrafiltration devices for
removal of unreacted fluorescent and isotope labels.
Method for Removal
of FITC from FITC-BSA
1. Bovine serum albumin was labeled with
fluorescein-isothiocyanate (FITC); the free
unincorporated label was not removed.
2. Two mL of FITC-labeled BSA solution at
0.5 mg/mL were loaded into two Amicon
Ultra-4 10K NMWL devices and centrifuged
at 3,000 x g for 10 minutes.
3. Retentates (about 50 µL each) were re-diluted
to 2 mL with water and centrifuged again.
This step was repeated twice.
4. After each ultrafiltratation, the retentate
and filtrate were analyzed by SDS-PAGE
and their fluorescence measured on a
SpectraFLUOR™ plate reader (Tecan) at
excitation 485 nm and emission 530 nm.
5. The SDS PAGE gel was scanned on a
Storm® (GE Healthcare) fluorescence scanner.
28
Results
Figure 8.1 shows the SDS-PAGE gel of FITC-labeled
BSA before and after each of four diafiltration
cycles. Unincorporated FITC is clearly visible in the
starting material and in the first filtrate. After only
one cycle of ultrafiltration, the majority of the free
label is removed. Subsequent cycles of filtration
result in BSA that is virtually free of unincorporated
FITC.
Fluorescence measurement offers a more sensitive
method to monitor the ultrafiltration process. Figure
8.2 shows the change in fluorescence of filtrate and
retentate during four cycles of FITC-BSA diafiltration.
The results indicate that almost 80% of free FITC
can be removed after the first ultrafiltration and
three rounds result in 98% removal. Therefore,
ultrafiltration can be used to clean up protein
labeling reactions as a viable alternative to gel
filtration. Each ultrafiltration using Amicon Ultra-4
devices is accomplished in 10 –15 minutes and
allows high recovery of target protein. While gel
filtration results in diluted protein fractions, ultra-
filtration offers the additional advantage of concen-
trating protein while removing unincorporated label.
Purification of Radiolabeled
Tumor Necrosis Factor
Courtesy of Mathew L. Thakur, Ph.D., Thomas
Jefferson University Hospital, Philadelphia, PA
Tumor necrosis factor (TNF) is a low-MW protein
(approximately 17 kDa) predominantly derived from
macrophages (TNF-alpha) or activated lymphocytes
(TNF-beta). Different cell types, normal and
malignant, possess receptors for TNF. The finding
that TNF can destroy tumors in vivo, even in the
absence of the direct lytic effect of neoplastic cells,
has led researchers to the hypothesis that TNF
 Figure 8.1
60,000
SDS-PAGE of FITC-labeled BSA
Lane 1: Starting material 50,000 Retentate
Lane 2: Retentate after first ultrafiltration

(Aribitrary Units)
Filtrate
40,000

Fluorescence
Lane 3: Retentate after 2 rounds of ultrafiltration
Lane 4: Retentate after 3 rounds of ultrafiltration 30,000
Lane 5: Retentate after 4 rounds of ultrafiltration
20,000
Lane 6: Filtrate after first ultrafiltration
Lane 7: Filtrate after 2 rounds of ultrafiltration 10,000
Lane 8: Filtrate after 3 rounds of ultrafiltration
0
Starting Spin 1 Spin 2 Spin 3 Spin 4
material

Figure 8.2 Fluorescence measurements of the filtrates and retentates after each
FITC-BSA of four cycles of FITC-labeled BSA ultrafiltration. Free label was transferred through
the 10,000 NMWL membrane while labeled BSA was retained and concentrated.
All retentates and filtrates were volume adjusted to 2 mL prior to the measurement.

FITC Table 8.1 Comparison of Centricon centrifugal device

and gel filtration for purification of Tc-99m TNF
1 2 3 4 5 6 7 8
Centricon Sephadex
Device Column
TNF Recovery 83 ± 2% 77.3 ± 8.5%
Time 70 minutes 40 minutes
labeled with short-lived gamma-emitting radio-
Volume 75 µL 9 mL
nuclides may be useful in scintigraphic imaging
of certain tumors.
In order to prepare radiolabeled TNF, ten
micrograms of purified TNF were labeled with
metastable isotope Tc-99m. Centricon 3K NMWL
microconcentrators and Sephadex™ G-25 gel
filtration columns were used to separate Tc-99m-TNF
from unbound Tc-99m. Human serum albumin and
lysozyme were added as a carrier in both cases.
The results of this experiment indicate that
Centricon 3K NMWL concentrators may be used
to purify proteins of small molecular weight from
unbound radioactivity without excessive loss of
proteins. When the quantity of such a protein is
limited, the addition of some carrier molecule after
radiolabeling may be necessary. While eliminating
more than 98% of the unbound radioactivity,
Centricon 3K NMWL units concentrate the protein
with high recovery and without loss, denaturation
or excessive dilution.

29
Protocols • Proteins

Urine Concentration with

Amicon Ultra Centrifugal Filters

Introduction Method
The measurement of specific proteins in urine is 1. Determine the total protein in a 24-hour urine
important for the diagnosis and management of specimen.
disease states. In most cases, the content of these 2. Fill Amicon Ultra-4 device with 4 mL of urine.
proteins in urine is too low to be detected and 3. Centrifuge at 3400 x g for 30 – 45 minutes
needs to be concentrated. Amicon Ultra-4 devices (approximately 25 – 50 µL concentrate volume).
can be used to concentrate urine samples prior to This produces up to a 160-fold increase in
clinical laboratory analyses. For example, patients concentration.
with multiple myeloma exhibit a proliferation of one
4. Insert a pipetter into the bottom of the filter
antibody-producing plasma cell, which leads to
unit and withdraw the concentrated sample.
excess production of free immunoglobulin light
5. Perform agarose electrophoresis on the
chains known as Bence-Jones proteins. After sample
concentrate to quantitate light chains and
enrichment in the Amicon Ultra-4 10K NMWL
identify other proteins. Determine the
device, immunofixation electrophoresis can be used
percentage of light chains with respect to
to identify free light chains (Bence-Jones proteins) in
the total number of components in the urine.
urine by forming a light chain-antibody complex.
Then multiply the percentage of light chains
Also, agarose electrophoresis can be used to
by the total 24-hour protein concentration
quantitate light chains and identify additional
(grams per 24-hour volume).
low molecular weight proteins such as albumin,
α-1 globulins, transferrin and IgG that can be 6. Perform immunofixation electrophoresis on
present in renal tubular disorders. Ultrafiltration of the concentrate to identify light chains.
urine samples in Amicon Ultra-4 devices provides
reproducible, high sample recovery for electro- Acknowledgements
phoretic analyses, usually in 45 minutes or less. Research using Amicon Ultra devices for urine
concentration in this protocol was conducted by
Materials Mark Merchant, Ph.D. at Helena Laboratories,
• Amicon Ultra-4 device, 4 mL, 10K NMWL Beaumont, TX.
• Centrifuge with fixed-angle or swinging bucket
rotor capable of 3400 x g
• Kit for microprotein determination (i.e. Sigma
# 610-A/Brilliant blue G/Coomassie™ blue)
• Pipetter with 200 µL tip
• Electrophoresis (agarose gel) and immunofixation
equipment with apparatus and reagents

30
References Additional Notes
1. Tietz N., Clinical Guide to Laboratory Tests, 1. Amicon Ultra devices can also be used to
2nd ed. Philadelphia:W.B. Saunders; concentrate serum, plasma and cerebrospinal
1990;362–363.
fluid for similar analyses. A concentration of
2. Kahns L, Clinical Chemistry 1991; approximately 20 mg/mL is required in order to
37:1557–1558.
detect free light chains from diseased patients
3. Cleveland Clinic homepage. Accessed by agarose electrophoresis. Detection by
July 2002. www.clevelandclinic.org/
immunofixation electrophoresis is 10 times more
myeloma/DiagnosisAndTreatmentOf
sensitive than by agarose electrophoresis.
MultipleMyeloma.html
2. Normal heterogeneous immunoglobulins may
4. Christenson RH, et al. Clinical Chemistry
1983;29(6):1028–1030. also be seen in urine concentrate with immuno-
fixation electrophoresis. This “ladder effect” is
5. Christenson RH, and Russell ME. Clinical
Chemistry 1985;31(6):973. comprised of microheterogenous light chains.
Bence-Jones proteins may be within this ladder.
To verify the presence of Bence-Jones proteins
requires additional analysis by two-dimensional
electrophoresis.
3. If there is excess antigen, dilution of the
concentrate will be required until equilibrium is
achieved between the antigen (Bence-Jones
protein) and the antibody.
4. Millipore also offers static concentrators
(Minicon® devices) for concentration of
Bence-Jones protein in urine.

31
Protocols • Proteins

Affinity Purification with

Ultrafree-MC Centrifugal Filters

Introduction Although the method is relatively simple, care must

be taken not to remove the chromatography resin
Affinity interaction chromatography is often the
when pipetting off the supernatant.
single most effective step in any protein purification
Pre-packed mini-spin columns are a convenient
procedure. Up to 95% purity can be achieved in
tool for small-scale protein purification. They are
one step, depending on the nature of the interaction
operated by centrifugation and usually require
and the starting composition of the protein solution.
less than an hour for the whole procedure.
Well known examples of highly specific affinity
Another alternative for small-scale purification
interactions include antibodies and protein A/G;
are centrifugal devices with microporous membrane,
multiple histidine tags and nickel; streptavidin and
such as Ultrafree-MC centrifugal devices. The
biotin; antibodies and antigens; and many others.
sample can be added to the filter basket and mixed
Less specific interactions are also used for enrich-
for the needed residence time and then centrifuged.
ment or depletion protocols, including albumin
The process removes the interstitial liquid but does
depletion on cibacron blue resin, glycoprotein
not dehydrate the beads. Washing and elution can
enrichment on concavalin A resin, and capture of
also be performed in a similar manner and are more
nucleic acid-binding proteins on heparin resin.
effective due to the efficient removal of buffer
Affinity chromatography is often employed in
and/or eluant.
the small-scale batch mode as a quick method for
Ultrafree-MC centrifugal filter units with
microgram-scale protein purification. The typical
microporous membrane (Figure 9.1) come with low
protocol involves:
protein-binding Durapore PVDF membrane in five
1. Pipetting a small volume of affinity resin into
different pore sizes from 0.1 to 5.0 µm. Affinity
a microfuge tube that contains the sample
resin can be loaded into the filter basket and the
2. Vortexing the tube for a few minutes
device used as a “home-made” mini-spin column.
3. Centrifuging the resin to the bottom We show the applicability of the device for
4. Pipetting off the supernatant purification of rabbit IgG on PROSEP-A resin and
5. Washing a few times (using steps 2 and 3) His-tagged C-RP protein on three different
6. Eluting with a small amount of eluant commercial metal-chelate resins.

Materials
• Ultrafree-MC 0.45 µm centrifugal devices
(Millipore cat. no. UFC3 0HV 00)
• PROSEP-A high capacity resin
(Millipore cat. no. 1131 118 26)
• Rabbit serum Gibco
(Invitrogen lot no. 1132782)
Figure 9.1
• Micro-centrifuge Biofuge® Pico
Ultrafree-MC
centrifugal (Heraeus instruments)
filter unit • Jouan CR1822 fixed angle rotor centrifuge

32
• Xcell SureLock™ Mini-cell vertical electrophoresis
system (Invitrogen cat. no. EI0001)
• NuPage® NOVEX Bis-Tris 4 –12%, 1 mm thick,
15 well SDS gels, (Invitrogen cat. no. NP0323)
• NuPage Sample Reducing agent (10X)
(Invitrogen cat. no. NP009)
• NuPage SDS Sample Buffer (4X)
(Invitrogen cat. no. NP007)
• SimplyBlue™ SafeStain Coomassie G-250 stain
(Invitrogen cat. no. LC6060)
Method for IgG Purification
Solutions
• PROSEP-A binding buffer A:
1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0
• PROSEP-A elution buffer B2:
0.2 M Glycine/HCl, pH 2.5
• PROSEP-A neutralization buffer:
1 M Tris/HCl, pH 9.0
Procedure
1. 200 mg of PROSEP-A media were placed in
Ultrafree-MC 0.45 µm filter basket.
2. The columns were equilibrated with 400 µL of
binding buffer A and centrifuged for 1 minute
at 100 x g.
3. 200 µL of rabbit serum were diluted 1:1
with binding buffer and the entire volume
was loaded into the spin column containing
PROSEP-A resin.
4. Devices were placed on a shaker for 15 minutes
at room temperature and centrifuged at 100 x g
for 5 minutes. Flow-through was collected for
future analysis.
5. Three consecutive washes of 400 µL each
were performed by adding 400 µL of binding
buffer A and centrifuging at 2,000 x g for
2 minutes each.
6. 200 µL of elution buffer B2 were added and
centrifuged for 2 minutes at 2,000 x g.
7. 26 µL of neutralization buffer were added to
each collection tube. A second elution was
collected after repeating the same process
one more time.
Method for His-tagged
C-RP Purification
Solutions
• Lysis buffer:
50 mM sodium phosphate, 300 mM sodium
chloride,10 mM immidazole, pH 7
• Binding buffer:
50 mM sodium phosphate, 300 mM sodium
chloride, 10 mM immidazole, pH 7
• Wash buffer:
50 mM sodium phosphate, 300 mM sodium
chloride, 20 mM immidazole, pH 7
• Elution buffer:
50 mM sodium phosphate, 300 mM sodium
chloride, 250 mM immidazole, pH 7
• 1 mg/mL Lysozyme stock
• Benzonase
Procedure
1. Recombinant proteins were expressed in
Escherichia coli.
2. Cells were prepared at a 10X concentration
using lysis buffer. Lysozyme was added to a
concentration of 0.1 mg/mL. To reduce the
viscosity, benzonase was added to the lysate.
The lysates were clarified by centrifugation.
3. 200 µL of the 50% resin slurry were added to
the Ultrafree-MC device and the residual fluid
was removed by centrifugation for 1 minute
at 500 x g.
4. The resin was equilibrated with 500 µL of
binding buffer and centrifuged for 2 minutes
at 500 x g.
5. 500 µL of the clarified lysate were added
to the resin.
6. The His-tagged proteins were bound
for 30 minutes with light agitation.
7. The lysate was removed by centrifugation
at 500 x g for 10 minutes.
8. The resin was washed with 500 µL of wash
buffer for 5 minutes with agitation. The wash
solution was removed by centrifugation for
5 minutes at 500 x g. This step was repeated
two more times.
9. 250 µL of elution buffer were added to the
Ultrafree-MC device and mixed for 5 minutes.
Purified protein was recovered by centrifugation
at 500 x g for 1 minute.
33
 1 2 3 4 5 6 7 8
Results
Figure 9.2 Rabbit IgG kDa
purification on PROSEP-A resin 200
The results of purifying rabbit IgG using Ultrafree-MC
in Ultrafree-MC devices. centrifugal devices are shown in Figure 9.2. The
116.3
Lane 1: Molecular weight 97.4 device was challenged with approximately 14 mg
66.3
standards of total protein, with an estimated IgG content of
55.4
Lane 2: Rabbit serum 1.5 – 2 mg. The original serum, flow-through, three
Lane 3: Flow through washes and two eluted fractions were analyzed by
Lanes 4 – 6: Three consecutive 36.5

washes
Lanes 7, 8: Eluted IgG from
two devices
Figure 9.3 His-tagged
protein purification using
Ultrafree-MC devices.
Lane 1: Molecular weight
31.0 SDS-PAGE. The total amount of purified IgG, as
21.5
estimated by OD280, was 1.2 mg and 1.1 mg
on two devices processed in parallel. The whole
14.4
procedure was completed in less than 1 hour.
6.0
This method can be useful for monitoring the titer
of antigen-specific antibodies after immune
activation, or whenever small amounts of IgG
need to be purified.
1 2 3 4 5 6 7 Figure 9.3 shows the results of His-tagged
kDa
200 protein purification in Ultrafree-MC devices using
Ni-NTA agarose and BD Talon™ resin. Two
116.3

standards
97.4 recombinant proteins were purified: C-RP, high-
66.3 RT66
Lane 2: E. coli lysate 55.4 expressing protein of 26 kDa; and RT66, low-
expressing C-RP expressing protein of 66 kDa. As seen in Figure 9.3,
protein 36.5 both purifications resulted in high purity proteins.
Lane 3: E. coli lysate 31.0

expressing RT66 C-RP The amount of proteins purified out of 500 µL of

21.5
protein lysate was 32 – 35 µg for C-RP protein and 8 µg
Lanes 4 , 5: Proteins purified 14.4 for RT66.
on Ni-NTA resin
The data show that affinity batch purification
Lanes 6, 7: Proteins purified 6.0
on BD Talon resin can be effectively performed on a small scale
using Ultrafree-MC devices loaded with resin.
The process combines the high efficiency of batch
binding and washing with the handling convenience
of a mini-spin column. With minimal hands-on time,
the method provides flexibility of resin to lysate
ratio and binding conditions, independent of
centrifugation speed and rotor angle. This method
is applicable to recombinant protein purification,
antibody purification and immunoprecipitation.

34
 Protocols • Proteins

Use of Centrifugal Filter Devices

as an Alternative to Stirred Cells

Introduction The eluted proteins were concentrated from

Stirred cell devices have been successfully used to
purify proteins from large volumes of solution for
many years. However, the need for assembly and
cleaning between uses, and the lengthy separation
times, can present difficulties for active, or under-
staffed, laboratories.
Millipore’s large volume centrifugal filter devices
come preassembled and ready to use. Individual
devices are available for processing 20 or 70 mL
volumes. For larger volumes, multiple devices can be
spun simultaneously. Spin times are typically
measured in minutes. And unlike stirred cells,
centrifugal devices run unattended. There is no need
to refill a reservoir and, therefore, less risk of
adventitious contamination.
The following protocol demonstrates the use
of Centricon Plus-70 centrifugal filter devices to
purify and concentrate proteins from large volumes
of solution.
volumes of 50 0 –1000 mL to 5 –10 mL using a
Centricon Plus-70 centrifugal filter device in a
swinging bucket rotor at 3000 x g for 10 minutes
(60 mL per filter unit). Buffer exchange was also
performed subsequently using the same units in
tissue culture grade sodium azide-free PBS. The
concentrated samples were then filter-sterilized
under sterile conditions using a 0.2 µm pore filter.
Results
Spectrophotometric evaluation of alpha-gamma
fusion protein recovery using Centricon Plus-70
devices showed a result of 18.8 mg/L of super-
natant. HPLC trace analysis revealed the chro-
matogram shown in Figure 10.1.
Acknowledgement
Protocol courtesy of GKT School of Biomedical
Sciences, London.

Abstract
This study aims to achieve purification and concen-
tration of a fusion protein composed of the alpha
140
and gamma subunits of the human high affinity IgE
120
receptor. The alpha-gamma fusion protein is purified
Absorbance Units

and then coupled to Sepharose® (GE) beads to 100

produce an affinity column for isolating human IgE 80
antibodies.
60

Method 40

The alpha and gamma subunit components of the 20

high affinity IgE receptor were transfected into 0

mouse myeloma cell line NS0 and the secreted 0 5 10 15 20 25 30

fusion protein was purified on a Protein G column. Time (min)

Figure 10.1 HPLC profile of alpha-gamma fusion component of the human FcεRI

35
Protocols • Nucleic Acids

Concentrating and Desalting DNA or RNA

with Microcon or Centricon Centrifugal Filters

Introduction which often denatures labile species. DNA and

RNA samples with starting concentrations as low as
Centrifugal filter devices serve as powerful tools in
5 ng/mL can be routinely concentrated in minutes
molecular biology applications, such as DNA or
with 99% recovery of starting material, and without
RNA concentration and desalting procedures.
the use of co-precipitants. Centrifugal concentrator
Ultrafiltration (UF) is a pressure-driven, convective
devices are ideal for separating high and low-
process that uses semipermeable membranes to
molecular weight species. Ultrafiltration can also
separate species by molecular size and shape.
be used to change solvents by diafiltration. In this
UF is highly efficient, allowing for concentration
process, the sample is concentrated, then diluted
and desalting at the same time. Unlike the use of
to the original volume with the desired buffer and
chemical precipitation methodologies (i.e., ethanol,
concentrated again, thus “washing out” the original
phenol/chloroform), there is no phase change,
solvent.
Millipore ultrafiltration membranes are character-
ized with well-defined, globular solutes (proteins).
Table 11.1 Nucleotide cut-off guidelines for Microcon and Centricon
centrifugal devices (based on >90% recovery of nucleic acids) Typically, the nominal molecular weight limit
(NMWL) for a membrane is the point at which over
Single-Stranded* Double-Stranded
NMWL Nucleotide Cut-off (bp) Nucleotide Cut-off (bp) 90% of a solute with that molecular weight will be
retained. To process DNA or RNA, the membrane
3K 10 10
needs to be characterized according to the number
10K 30 20
of nucleotides in the fragment. Polynucleotides (DNA
30K 60 50 and RNA) have tertiary structures that are ordinarily
50K 125 100 more extended than those of typical globular
100K 300 125 proteins of similar size. Millipore has determined
nucleotide cut-offs (based on the number of bases
*Single-stranded nucleic acids with extensive secondary structure will be better
retained than those without.
or base pairs in a fragment of DNA or RNA) that
correspond to the NMWL of each of their low
binding membranes. The nucleotide cut-off (NCO)
indicates the fragment length of single- or double-
Table 11.2 Desalting and recovery with Centricon 30K NMWL devices
stranded DNA or RNA that one would expect to
Spin Number % DNA Recovered % CsCl Removed recover at 90% efficiency with a unit of the named
1 94 91.5 NCO. It is best to choose the NCO with about half
2 91 98.9 the length of the fragment of interest. For example,
3 96 99.9 selecting a 30K NMWL membrane for a 50 base
pair fragment generally results in 90% product
Using 1 mL of 25 µg/mL E. coli DNA in 6 M CsCl, sample was repeatedly
recovery. A 10K NMWL membrane would provide
concentrated to 0.1 mL. Sample spun at 2000 x g in a 45 degree fixed angle rotor.
The concentrated sample was reconstituted to original 1 mL volume by adding
for closer to 100% recoveries but would take much
50 mm Tris. After three spins, CsCl concentration was reduced by four orders of longer to process the sample. For nucleic acid
magnitude. Equivalent results can be obtained with the Microcon 30K NMWL filter samples >500 base pairs, a 100K NMWL
unit (as well as other NMWLs). membrane is appropriate.

36
 Table 11.1 offers guidelines for DNA/RNA
retention based on the nucleotide content of single-
and double-stranded pieces. For example, more
than 90% of a single-stranded 30-mer will typically
be retained by a Microcon 10K NMWL or a
Centricon 10K NMWL centrifugal filter device.
Concentrating dilute DNA solutions is a key step
for many subsequent preparative and analytical
procedures. For example, standard plasmid
preparations involving cesium chloride, equilibrium
centrifugation, and gel filtration yield DNA in large
volumes that require concentrating prior to precipita-
tion. DNA concentration is also necessary in
purifying restriction fragments from gels.
There are three major techniques currently
available for concentrating nucleic acids:
• repeated extractions with n-butanol
• adsorption to ion exchange resin, followed
by high salt elution
• lyophilization
The first method has the disadvantage that
n-butanol concentrates all solutes, including salt,
which tends to co-precipitate with DNA upon
addition of ethanol. The second method, ion
exchange, aside from requiring buffers of various
ionic strengths, yields DNA in a high salt solution.
Figure 11.1 shows that both recovery and
biological activity are similar when DNA is concen-
trated with Millipore’s high recovery centrifugal filter
devices versus conventional techniques. However,
the efficiency and immediate capability to further
utilize the nucleic acids without a subsequent
desalting step is a significant advantage when
using Millipore centrifugal devices.
Methods
Microcon Device
1. Select a Microcon unit with nucleotide cut-off
equal to or smaller than the molecular size
of the nucleic acid you want to retain (refer
to Table 11.1).
2. Insert Microcon sample reservoir into one of the
two vials provided for each unit.
3. To concentrate (without affecting salt concen-
tration): Pipette up to 500 µL of DNA or RNA
sample into the reservoir. Spin for recommended
time, not exceeding recommended g-force
guidelines shown in Table 11.3.
Table 11.3 Recommended g-force and spin time for Microcon devices

The third method, lyophilization, increases the Maximum Spin Time Spin Time
G-Force (min.) (min.)
concentration of buffer components, which can
NMWL Rating at 4 °C at 25 °C
result in degradation of nucleic acids.
3K 14,000 185 95
Ultrafiltration membranes retain DNA or RNA but
are permeable by smaller ionic buffer components. 10K 14,000 50 35
Ultrafiltration alone does not change buffer com- 30K 14,000 15 8
position. The salt concentration in a sample 50K 14,000 10 6
concentrated by Microcon or Centricon centrifugal 100K 500 25 15
filter devices will be the same as in the original
sample. For desalting, the concentrated sample is
diluted with water or buffer to its original volume Figure 11.1 Retention of labeled DNA fragments 1 2 3
and spun again in a process called diafiltration. by Centricon 10K and 30K NMWL devices.
1631
This removes the salt by the concentration factor of Two 1 µL aliquots of 32P-labeled pBR322 DNA
the ultrafiltration. fragments were diluted to 1.0 mL using TE buffer
For example, if a 500 µL sample containing (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). They
were subsequently concentrated to 40 µL with the
100 mM salt is concentrated to 25 µL (20X
Centricon devices. 20 µL of each concentrate were 517/506
concentration factor), 95% of the total salt in the analyzed on a 2.5% agarose gel. The intensities of 396
sample will be removed. The salt concentration in the autoradiographic bands were compared to bands 344
298
the sample will remain at 100 mM. Rediluting the from a 1 µL aliquot of the solution of labeled DNA
221/220
sample to 500 µL will bring the salt concentration fragments that had not been concentrated by the
devices. The gel was dried and autoradiographed. 154
to 5 mM. Concentrating to 25 µL once more will
remove 99% of the original total salt. The concen- Lane 1: Not concentrated using Centricon device
75
trated sample will now be in 5 mM salt. For more Lane 2: Concentrated with Centricon 10K NMWL device
Lane 3: Concentrated with Centricon 30K NMWL device
complete salt removal, an additional redilution and
spinning cycle will remove 99.9% of the initial
salt content (see Table 11.2).

37
 4. To exchange salt: Add the proper amount of Centricon Device
appropriate the diluent to bring the concentrated 1. Select a Centricon unit with a nucleotide cut-off
sample to 500 µL. Spin for the recommended equal to or smaller than the molecular size of
time, not exceeding g-force shown in Table 11.3. the nucleic acid you want to retain (refer to
To achieve lower salt concentration, repeat the Table 11.1).
entire step as necessary. NOTE: Do not let
2. To concentrate (without affecting salt concentra-
filtrate vial overfill.
tion): Pipette up to 2 mL of DNA or RNA sample
5. Remove reservoir from vial and invert into a new into the reservoir. Spin for the recommended time,
vial (save filtrate until sample has been analyzed). not exceeding g-force shown in Table 11.4.
6. Spin for 2 minutes at 500 –1000 x g to recover 3. To exchange salt: Add the proper amount of
nucleic acid in the vial. appropriate the diluent to bring the concentrated
7. Remove reservoir. Cap vial to store. sample to 2 mL. Spin for the recommended time,
not exceeding g-force shown in the Table 11.4.
To achieve lower salt concentration, repeat the
Table 11.4 Recommended g-force and spin time for Centricon devices entire step as necessary. NOTE: Do not let
Maximum Spin Time (min.) filtrate vial overfill.
NMWL G-Force Rating at 25 °C
4. Remove reservoir from filtrate vial and invert unit
3K 7,500 120 (save filtrate until sample has been analyzed).
10K 5,000 60 5. Spin for 2 minutes at 300 –1000 x g to recover
30K 5,000 30 nucleic acid in the retentate vial.
50K 5,000 15 6. Remove reservoir. Cap retentate vial to store.
100K 1,000 30

38
 Protocols • Nucleic Acids

Preparing Samples for Forensics Identification

Analysis with Microcon Centrifugal Filters
Centrifugal filter devices with ultrafiltration (UF) remove inhibitors that can adversely affect PCR
membranes are used by forensics laboratories to amplification. In addition, Chelex extraction of small
isolate genomic DNA for human identification. The bloodstain samples often results in DNA concentra-
source materials are typically blood stains and/or tions below detection limits. Concentrating extracted
other bodily fluids obtained from crime scenes, samples with Microcon 100K NMWL devices
criminal suspects, or human remains. improves the success rate of STR amplification and
The isolated genomic DNA is used to identify yields a full STR allele profile even with minute blood
individual persons based on their patterns of Short or semen stains. According to the New York City
Tandem Repeats. STRs are polymorphic DNA loci Department of Forensic Biology, several of the peaks
that contain repeated DNA sequences. Because the shown in Figure 12.1 would have been below the
repeats can vary from two to seven bases in length, detection threshold and identification would have
many different alleles are possible for each locus. been negative or inconclusive without sample
The parallel analysis of 9 to 13 polymorphic cleanup using Microcon devices.
STR loci can unequivocally identify an individual.
Several suppliers offer STR assay kits. Each Figure 12.1
supplier offers several versions of their kits with Genotype analysis of a
different protocols, modes of operation, and semen sample before
detection systems to suite the user’s needs. and after concentration
by a Microcon
Millipore’s UF-based centrifugal devices are
centrifugal filter.
specified in several of these protocols (AmpFLSTR® Electropherogram A
Profiler, Applied Biosystems; GenePrint® STR and shows a partial profile
GenePrint Fluorescent STR Systems, Promega). with a high molecular
weight loci in each
color missing.
Use of UF-based Centrifugal
Electropherogram B
Devices in New York City shows a full profile and
Depending on the type of evidence under investiga- higher peak heights.
A
tion, the Department of Forensic Biology in New Amounts amplified were
0.62 ng for electro-
York City had been preparing genomic DNA by
pherogram A and 1 ng
either (1) organic extraction (phenol/chloroform/ for electropherogram B.
isoamylalcohol) followed by alcohol precipitation or The sample was
(2) direct lysis in the presence of Chelex® beads extracted with Chelex
(Bio-Rad) with no further purification. beads. The peaks are
labeled with allele
The Department of Forensic Biology’s laboratory
name, size in base
has replaced the ethanol precipitation step with
pairs, and fluorescent
Microcon centrifugal filters with a 100K NMWL peak height. The
ultrafiltration membrane. The centrifugal devices improvement in results
provide a faster way to concentrate the purified is due not only to the
DNA without the use of toxic chemicals. increased DNA input
but also to the
The laboratory also uses the Microcon
concentration step
100K NMWL device after Chelex extraction to B following extraction.

39
Protocols • Nucleic Acids

PCR Purification with

Montage PCR Filter Units

Introduction Primer DNA

Salt
After polymerase chain reaction (PCR)1, amplified dNTP

DNA must be separated from excess reaction

components that can interfere with subsequent
manipulations such as cloning or sequencing.
The Montage PCR filter unit is a single-use device
that simplifies the purification of PCR products.
It concentrates amplified DNA and removes primers
and unincorporated dNTPs, while providing
excellent capacity, high recovery, and high purity. Step 1 Step 2 Step 3
The method is quick and highly reproducible, which
makes it ideal for processing one-up or multiple
samples in middle-throughput operations. 5. Add 20 µL purified water or TE buffer to sample
reservoir.
Materials 6. Invert the reservoir and spin at 1000 x g for
• Variable speed microcentrifuge 2 minutes to retrieve the purified PCR (step 3).
• Montage PCR filter unit
• Purified water (such as Milli-Q water) or TE buffer Results
• PCR reaction (aqueous phase) Montage PCR filter units were used to purify 100 µL
PCR reactions according to the specified protocol.
Procedure After recovering the DNA fragments (n = 10) using
1. Insert the Montage PCR sample reservoir into a reverse spin, the samples were separated by
one of the two vials provided. agarose gel electrophoresis. Recoveries of the
2. Fill the reservoir with 300 µL purified water or TE various PCR products were determined by
buffer. Add 100 µL PCR reaction to the reservoir densitometry (Figure 13.1).
(step 1). Smaller volumes of PCR product may The Montage PCR filter unit is a convenient
be used, but the volume should be adjusted to a method for single-sample PCR purification. The
final volume of 400 µL. high-performance device purifies PCR products in a
single centrifugation step. Purified samples are ready
3. Spin the Montage PCR device at 1000 x g
for downstream applications with no additional
for 15 minutes (step 2). NOTE: For optimal
purification steps.
recovery, do not centrifuge longer than the
specified 15 minutes or greater than 1000 x g.
References
4. To recover DNA, remove the sample reservoir
1. PCR is covered by U.S. patents issued to
from filtrate collection vial and place in a
Hoffmann-LaRoche, Inc.
clean vial.

40
 100%

80%
Percent Recovery

60%

40%

20%

0%
137 bp 301 bp 500 bp 657 bp 1159 bp

Figure 13.1 Purification of PCR products using Montage PCR Filter Units

Figure 13.2 Typical electropherogram shows an 1159 bp PCR product purified with a Montage PCR filter unit.
Note the uniform signal intensity and long read lengths. Purified samples are ready for cloning or sequencing with
no additional purification steps.

41
Protocols • Nucleic Acids
Preparation of Fluorescent DNA Probe from Human
mRNA or Total RNA with Microcon Centrifugal Filters
Introduction
Microarrays are efficient tools that enable the high
throughput identification of genes that are differen-
tially regulated in response to disease, drugs or
other stimuli. With the completion of several key
genome sequencing projects, scientists now have
the ability to custom design DNA microarrays
specific to their research interests. The recent
advances in robotics, bioinformatics and detection
technologies have greatly simplified the manufacture
and analysis of microarrays. However, the successful
application of microarray technology requires highly
purified, fluorescently labeled cDNA probes. The
application of ultrafiltration technology to this
challenge has resulted in a robust, efficient and
rapid method for the generation of high quality
fluorescently labeled probes suitable for use with
microarrays. The protocol below describes a
method for the generation and purification of
fluorescently labeled probes using a Microcon
30K NMWL centrifugal filter device.
42
Method
1. To anneal primer, mix 2 µg of mRNA or
50 –100 µg total RNA with 4 µg of a regular
or anchored oligo-dT primer in a total volume
of 15.4 µL as shown in Table 14.1.
2. Heat to 65 °C for 10 minutes and cool on ice.
3. Add 14.6 µL of reaction mixture each to Cy3
and Cy5 reactions as shown in Table 14.2.
4. Incubate at 42 °C for 1 hour.
5. Add 1 µL SSII (RT booster) to each sample.
Incubate for an additional 0.5 –1 hours.
6. Degrade RNA and stop reaction by addition
15 µL of 0.1 N NaOH, 2 mM EDTA and
incubate at 65 – 70 °C for 10 minutes.
If starting with total RNA, degrade for
30 minutes instead of 10 minutes.
7. Neutralize by addition of 15 µL of 0.1N HCl.
8. Add 380 µL of TE (10 mM Tris, 1 mM EDTA)
to a Microcon 30K NMWL device column.
Next add the 60 µL of Cy5 probe and the
60 µL of Cy3 probe to the same Microcon
device. NOTE: If re-purification of Cy dye
flow-through is desired, do not combine
probes until Wash 2.
9. Wash 1: Spin column for 7 – 8 minutes at
14,000 x g.
10. Wash 2: Remove flow-through and add
450 µL TE and spin for 7– 8 minutes at
14,000 x g. It is a good idea to save the
flow trough for each set of reactions in a
separate microcentrifuge tube.
11. Wash 3: Remove flow-through and add
450 µL 1X TE, 20 µg of Cot1 human DNA
(20 µg/µL, Gibco-BRL), 20 µg polyA RNA
(10 µg/µL, Sigma, #P9403) and 20 µg tRNA
(10 µg/µL, Gibco-BRL, #15401-011).
Spin 7–10 min. at 14,000 x g. Look for
concentration of the probe in the Microcon
device. The probe usually has a purple color
at this point. Concentrate to a volume of
less than or equal to the volume listed in
the “Probe and TE” column in Table 14.3.
These low volumes are attained after the
center of the membrane is dry and the probe
forms a ring of liquid at the edges of the
membrane. Make sure not to dry the
membrane completely.
12. Invert the Microcon device into a clean tube
and spin briefly at 14,000 RPM to recover
the probe.
13. Select the appropriate row from Table 14.3.
Adjust the probe volume to the value indicated
in the “Probe and TE” column.
14. For final probe preparation add 4.25 µL 20X
SSC and 0.75 µL 10% SDS. When adding
the SDS, be sure to wipe the pipette tip with
clean, gloved fingers to remove excess SDS.
Avoid introducing bubbles and never vortex
after adding SDS.
The probe is now ready for hybridization.
Acknowledgements
Protocol courtesy of Patrick Brown, Max Diehn,
and Ash Alizadeh, Stanford University School
of Medicine

Table 14.1 Preparation of primer

Primer Cy3 Cy5 Notes
mRNA (1 µg/µL) x µL y µL 2 µg of each if mRNA; 50 –100 µg if total RNA
Oligo-dT (4 µg/µL) 1 µL 1 µL Anchored: 5'-TTT TTT TTT TTT TTT TTT TTV N-3'
Purified H2O (DEPC) to 15.4 µL to 15.4 µL
Total volume 15.4 µL 15.4 µL

Table 14.2 Preparation of probe

Final
Reaction Mixture Volume Unlabeled dNTPs Volume Concentration
5X first-strand buffer* 6.0 µL dATP (100 mM) 25 µL 25 mM
0.1 M DTT 3.0 µL dCTP (100 mM) 25 µL 25 mM
Unlabeled dNTPs 0.6 µL dGTP (100 mM) 25 µL 25 mM
Cy3 or Cy5 (1 mM, Amersham) 3.0 µL dTTP (100 mM) 10 µL 10 mM
Superscript® II (200 U/µL, Gibco-BRL) 2.0 µL Purified H2O 15 µL
Total volume 14.6 µL Total volume 100 µL

*5X first-strand buffer: 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2

Table 14.3 Final probe preparation

Cover Slip Size Total Hybrid Probe and TE 20X SSC* 10% SDS
(mm) Volume (µL) (µL) (µL) (µL)
22 x 22 15 12 2.55 0.45
22 x 40 25 20 4.25 0.75
22 x 60 35 28 5.95 1.05

*20X SSC: 3.0 M NaCl, 300 mM NaCitrate (pH 7.0)

43
Protocols • Nucleic Acids
Purification of In Vitro Synthesized mRNA with
Microcon or Centricon Centrifugal Filters
Introduction
In vitro transcription reactions employing T3, T7 or
SP6 phage-encoded RNA polymerases are widely
used to synthesize RNA from recombinant vectors
containing appropriate promoters. Production of
large amounts of specific RNA is valuable in the
preparation of hybridization probes and in vitro
translation studies; in the synthesis of ribozymes,
rRNA, SRP, antisense RNA and substrates for RNA
splicing; and in RNA-protein interaction studies.
Centricon and Microcon centrifugal filters are
well suited for the purification of radiolabeled RNA
transcripts1. Ultrafiltration can simultaneously and
efficiently remove unincorporated ribonucleotides
and salts from the transcripts and concentrate the
RNA. RNA molecules retain their integrity and are
recovered with high yields.
Purity of a transcript is especially important when
it is used in in vitro translation systems. Trace amounts
of ethanol, phenol, salts or excess cap analog used
during the synthesis of capped mRNA can cause a
dramatic decrease in translation efficiency.
After the transcription reaction is complete,
template DNA is usually degraded by the addition
of DNase I. The RNA is purified by two phenol/
chloroform extractions followed by ethanol pre-
cipitation. Other, less popular methods are gel
purification (used predominantly when separation
of full-length transcript from shorter RNAs is
important, e.g., ribonuclease protection assays)
or LiCl precipitation.
A series of experiments was performed in our
laboratory to determine the effectiveness of using
Centricon and Microcon devices to purify in vitro
synthesized mRNA and in vitro translation studies.
Results indicate that ultrafiltration can efficiently
remove inhibitory contaminants from mRNA
preparations, leading to increased translational
efficiencies.
44
Methods
RNA Transcription
For our studies we chose plasmid pGEM-luc
containing the luciferase gene (luc) in the center of
a multiple cloning cassette of the pGEM-11Zf (-)
plasmid (Promega). DNA template was linearized
with XhoI, followed by enzyme and salt removal by
diafiltration in Microcon 100K NMWL devices.
Linearized template was transcribed, using
MEGAscript® kit (Ambion) according to the recom-
mended protocol. After the reaction was completed
(3 to 4 hours), template DNA was degraded with
DNase I and the reaction mix added to a Microcon
30K NMWL device filled with 450 µL of water.
The device was spun for 20 minutes at 12,000 x g
in a temperature-controlled centrifuge at 4 °C.
Purified, concentrated RNA was recovered by
inverted spin. For the preparation of capped
transcript, cap analog m7G (5’) ppp (5’) G
(New England Biolabs, Inc.) was included in the
transcription reaction and the level of GTP reduced
(4:1 ratio of cap analog to GTP). To purify the
transcript by phenol/chloroform extraction, the
reaction mix was diluted with water and a one-tenth
volume of ammonium acetate stop solution was
added. The mixture was extracted once with
phenol/chloroform, followed by chloroform
extraction. RNA was precipitated with isopropanol
and the pellet resuspended in distilled water.
Alternatively, LiCl precipitation solution (one-half
volume) was added to the reaction mix, followed
by incubation at minus 20 °C for 1 hour. RNA was
pelleted by centrifugation and dissolved in water.
Size and integrity of the in vitro transcription products
were assessed by running an aliquot of the purified
RNA transcript on a formaldehyde/formamide
agarose gel. Ethidium bromide was added to the
RNA before lading on the gel to stain the RNA
sample and keep background fluorescence low2.
Translation In Vitro References
In vitro translations were performed in the Flexi™ 1. Krowczynska AM. bioSolutions 1993;2(1):1–2.
Rabbit Reticulate Lysate System (Promega) according 2. Ogretmen B, Ratajczak H, Kats A, Stark BC.
to standard luciferase RNA translation conditions Biotechniques 1993;14(6):932-5.
with minor modifications (Rnasin Ribonuclease 3. Polayes D. Focus 1991;13(4):130 –2.
inhibitor was omitted and 35S-methionine added). 4. Dasso MC, Jackson RJ. Nucl. Acid. Res.
Results of translation were analyzed by determina- 1989;17:3129.
tion of percent incorporation of 35S-methionine and
fold stimulation, compared to controls without RNA.
Minimum acceptable stimulation was 8-fold.
1 2 3 4 5
Figure 15.1 Comparison of pGem-luc transcript
Results purification methods. Transcripts were synthesized in
20 µL reactions. After DNase I treatment, RNA was
Aliquots of RNA transcript purified by different
purified from the reaction mix. 500 ng of purified
methods (ultrafiltration, phenol extraction and RNA were run on 1% agarose/formaldehyde gel.
LiCl precipitation) were run on a denaturing
Lane 1: 0.16 –1.77 kb RNA ladder (Gibco BRL)
agarose/formaldehyde gel. Results are shown in
Lane 2: RNA purified in Microcon 30K NMWL device
Figure 15.1. The banding pattern of the 1.7kb RNA
Lane 3: RNA purified in Centricon 100K NMWL device
transcripts is identical regardless of purification
Lane 4: RNA purified by phenol extraction
method. Similar results were obtained in the case of
Lane 5: RNA purified by LiCl precipitation
capped transcript (results not shown).
The effect of increasing the mRNA concentration
on the translational efficiencies was examined.
At low mRNA levels, the capped luc mRNA was Figure 15.2 Effect of
pGem-luc RNA concen- 140
translated three times more efficiently than the
tration on in vitro trans- 120
uncapped mRNA (Figure 15.2). At higher mRNA lation. Increasing amounts
100
levels, the translation of both transcripts was of uncapped Luc RNA
cpm x 104

80
comparable. Similar behavior was observed with transcripts and capped
CAT mRNA3. Even relatively high levels of mRNA Luc RNA were used in 60
Capped pGem-luc RNA

did not cause the decrease in translational efficien- translation reactions. 40 pGem-luc RNA
Incorporation of
cies noted by other groups4. This result could be 35S-methionine was
20

attributed in part to the lack of inhibitory contami- determined by TCA

0
0 0.5 1.0 1.5 2.0
nants in the mRNA preparation. precipitation. Both RNA µg RNA/50 µL Reaction
We also checked the effect of the RNA transcripts were purified
clean-up method on in vitro translational efficiency. with Microcon 30K
NMWL devices.
For details of various procedures, see the Methods
section. RNA purified by each of the methods
(1 µg) was translated and results showing total
35S-methionine incorporation are presented in
Table 15.1 Effects of RNA clean-up method on translation efficiency
Table 15.1. While there were no observable 35S-methionine
RNA Total Incorporation
differences between these RNAs by gel electro-
RNA 1 972,000
phoresis analysis (Figure 15.1), RNA purified by
ultrafiltration gave twice the translation efficiencies RNA 2 514,000
of phenol-extracted RNA. RNA 3 776,000

The RNA transcripts were cleaned using DNase I incubation followed by purification
in Centricon 100K NMWL devices (RNA 1), phenol/chloroform extraction (RNA 2),
or precipitation with 7.5 M LiCl (RNA 3). 1 µg of each RNA was used to program
50 µL in vitro translation reaction, using the Flexi Rabbit Reticulocyte Lysate System.
35S-methionine incorporation was determined by TCA precipitation. Data show the

average of three independent experiments.

45
Protocols • Nucleic Acids

Quantitative Recoveries of Nanogram Amounts

of Nucleic Acids with Microcon Centrifugal Filters

Introduction were added to each tube. The content was well

mixed and incubated for the specified period of
Molecular cloning experiments often require
time at –20 °C or –70 °C (dry ice). The solutions
concentration of nanogram quantities of DNA.
were then centrifuged at 12,000 x g in a fixed-
When dealing with such small amounts of nucleic
angle microcentrifuge at 4 °C for 15 minutes. The
acids, the use of coprecipitants (tRNA or purified
supernatants were carefully removed and pellets
glycogen) is essential for effective DNA recovery1.
resuspended in 50 µL of TE buffer. The radioactivity
However, when carrier tRNA cannot be used (for
in the precipitates and supernatants was then
example, when DNA is to be labeled with 32P dATP
determined by counting Cherenkov radiation.
and polynucleotide kinase), the only method of
The percentage of recovered DNA was calculated
recovering DNA as a precipitate in ethanol has
from precipitates and the initial counts. Each data
been the ultracentrifugation method developed by
point in the tables represents the average of at least
Shapiro2. Ultrafiltration, which has been traditionally
three samples.
used to concentrate and desalt protein samples
To precipitate the oligomer, 60 µL of TE buffer
simultaneously, can be an efficient alternative to
containing a given amount of 25-mer and 5 ng of
ethanol precipitation.
radiolabeled tracer was supplemented with 240 µL
In this experiment, three popular ethanol precipi-
of 5 M ammonium acetate and 750 µL of EtOH.
tation protocols for DNA and oligonucleotides are
Subsequent steps were identical to those described
compared with ultrafiltration in Centricon and
for DNA.
Microcon centrifugal filter devices. For a detailed
discussion on the effects of incubation time, Ultrafiltration
temperature and centrifugation parameters on
A solution of DNA (500 µL) was placed into a
ethanol precipitation, see Reference 3.
Microcon 30K NMWL unit and spun at 12,000 x g
for 10 minutes. A 500 µL solution of oligonucleo-
Methods tides in TE buffer was placed into a Microcon 3K
Plasmid pBR322 was digested with EcoR I and the NMWL unit and spun for 45 minutes at 12,000 x g.
3’ recessed termini were filled-in with alpha 32P The retentates were recovered by inverting the units
dATP, using Klenow fragment of DNA polymerase I. and centrifuging at 500 –1000 x g for 2 minutes.
A 25 nucleotide mixed base oligomer was radio- While concentrating the oligomer samples, it is
labeled by phosphorylation with bacteriophage T4 important to avoid high salt concentrations which
polynucleotide kinase. promote binding of single-stranded nucleic acids
to the cellulose-based ultrafiltration membrane.
Ethanol Precipitation
The radioactivity in the retentates and filtrates
All DNA precipitations were performed in 250 µL
was determined by counting Cherenkov radiation.
volume. Each tube contained a given amount of
Data represent averages of six samples each.
DNA, supplemented with 10 ng of radioactively
labeled pBR322 in 0.3 M sodium acetate buffer.
To precipitate the DNA, 2.5 volumes of 95% EtOH

46
Results can significantly improve recovery. In contrast to
EtOH precipitation, ultrafiltration using Microcon or
Standard methods for ethanol precipitation of
Centricon devices delivered almost 100% recovery
nucleic acids and ultrafiltration are compared in
with concentrations as low as 10 ng/mL and took
Tables 16.1 and 16.2. Use of Centricon 30K
only 10 minutes. The ultrafiltration devices were
NMWL and Centricon 3K NMWL (data not shown)
found to concentrate and desalt nucleic acids
devices gave results similar to those obtained with
effectively in one step resulting in high recoveries
Microcon devices. Recovery of DNA was assessed
and providing a quick, alternative to ethanol
at close to 100%, indicating that no significant
precipitation.
amount of nucleic acid was lost to the membrane
or device due to adsorption.
References
In contrast, DNA recovery using EtOH precipi-
1. Wallace DM. Precipitation of Nucleic Acids.
tation varied from as little as 14% as in the case
Methods in Enzymology 1987;152:41–8.
of DNA (10 ng/mL) precipitated for 15 minutes
2. Shapiro DJ. Quantitative Ethanol Precipitation
at – 70 °C to a maximum of 76% after overnight
of Nanogram Quantities of DNA and RNA.
incubation at – 20 °C. Anal Biochem 1981;110:229–31.
3. Crouse J, Amorese D. Ethanol Precipitation:
Discussion Ammonium Acetate as an Alternative to
Poor recovery of nucleic acids at very low concen- Sodium Acetate. Focus 1987;9(2):3–5.
tration with ethanol precipitation may be partially
due to the fact that small amounts of DNA do not
adhere well to the tube walls following sedimenta-
tion unless high g forces (ultracentrifugation) are
employed. The yield of DNA incubated at – 70 °C
is slightly reduced, in agreement with previous
studies. Also an overnight precipitation at – 20 °C

Table 16.1 Efficiency of different methods for concentration of DNA

DNA Concentration
10 ng/mL 25 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL
Method % Recovery % Recovery % Recovery % Recovery % Recovery
EtOH, –70 °C, 15 min 14 15 23 52 55
EtOH, –20 °C, 30 min 13 20 25 60 72
EtOH, –20 °C, 18 hr 31 45 45 76 67
Microcon 30K NMWL Device 95 95 98 98 99

Table 16.2 Efficiency of different methods for concentration of oligonucleotides

Oligomer Concentration
10 ng/mL 25 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL
Method % Recovery % Recovery % Recovery % Recovery % Recovery
EtOH, –70 °C, 15 min 4 4 5 6 33
EtOH, –20 °C, 30 min 6 5 6 4 33
EtOH, –20 °C, 18 hr 17 20 15 30 58
Microcon 3K NMWL Device 93 94 93 95 95

47
Protocols • Nucleic Acids
Effect of Centrifugal Ultrafiltration on
Large Fragment DNA Integrity
Introduction
Centrifugal ultrafiltration provides a fast and easy
method for the concentration and desalting of
biological molecules. Previous reports document
the use of ultrafiltration for desalting samples of
nucleic acids1, removing excess primers from PCR2
reactions3, or concentrating DNA/RNA samples
without the need to use ethanol precipitation4.
Centricon and Microcon filters offer a
convenient, reproducible means for centrifugal
ultrafiltration of samples from 50 µL to 2 mL. They
insure high sample recovery with their patented
inverted recovery spin.
The analysis of complex genomes depends on
the ability of the researcher to prepare pure, high
molecular weight DNA. When preparing high
molecular weight DNA samples for cosmid cloning
or other applications, it is important to treat the
DNA gently to avoid shearing or other damage
to the sample. As virtually all protocols for the
preparation of high molecular weight DNA require,
at some point, buffer exchange and/or concentra-
tion, centrifugal ultrafiltration can provide an
efficient alternative to standard procedures.
1 2 3 4
Figure 17.1 Supercoiled DNA Ladder.
Lane 1: Starting material
Lanes 2, 3: Retentate from DNA ladder spun 3 times
at 12,000 x g in Microcon 30K NMWL
device
Lane 4: Retentate from DNA ladder spun 3 times
at 5,000 x g in Centricon 30K NMWL
device
48
To be useful, centrifugation must not cause
breakage of the DNA during the ultrafiltration
procedure. This article summarizes the results of a
series of experiments designed to evaluate the effect
of g forces on various samples of high molecular
weight DNA during centrifugal ultrafiltration in
Microcon or Centricon units.
Methods
DNA Ladder
As a model system to check for the introduction
of single-strand nicks during the ultrafiltration spin,
supercoiled Ladder DNA (Gibco-BRL, Gaithersburg,
MD) which ranges in size from 2 to 8 kb was used.
DNA diluted with TE buffer was loaded into
Microcon 30K NMWL and Centricon 30K NMWL
devices. The Microcon units were spun for 7 minutes
at 12,000 x g, and the Centricon units were spun
for 30 minutes at 5,000 x g. The concentrated
DNA sample (retentate) was diluted again with
TE buffer (to 500 µL or 2 mL, depending on the
device used) and spun once more, as described
above. The dilution step was repeated a third time.
After the third concentration spin, the retentate was
collected by placing the sample reservoirs upside
down in new vials and spinning the units for
1 minute at 1,000 x g.
The concentrated DNA was run on a 0.9%
SeaKem® GTG agarose gel (FMC) and stained
with ethidium bromide to monitor sample integrity
(Figure 17.1). DNA bands in lane 1 (starting
material) are indistinguishable from the bands on
lanes 2– 4, which were spun repeatedly in the
ultrafiltration devices. There is no evidence that any
supercoiled DNA was converted to the relaxed or
linear forms during the concentration procedure,
which would be the case had single-strand nicks
been introduced during centrifugation.
Discrete Size Plasmids
The next set of experiments monitored the effect of
centrifugal ultrafiltration on single population,
discrete size plasmids. The plasmids used were
pBR322 (4,361 bp; New England Biolabs, Inc.),
pSPT18 (3,104 bp; Boehringer Mannheim) and
pXTl (10,400 bp; Stratagene). Samples (1 µg) of
each plasmid were spun in Microcon 30K NMWL
or Centricon 30K NMWL units, as described
previously. The starting material and retentates
were run on a 1% agarose gel and stained with
ethidium bromide.
The results are shown in Figure 17.2. As in the
case of the DNA Ladder, the concentrated samples
(lanes 2, 3, 5, 6, 8, and 9) appear to be identical
with their corresponding starting material. Again,
there is no evidence of conversion of the supercoiled
form to the relaxed form after exposure to g forces
of 5,000 x g for 90 minutes and 12,000 x g for
21 minutes during the concentration spins.
Genomic Size DNA
To monitor the integrity of molecules in the size range
of genomic DNA after centrifugation, lambda DNA
(49 kb; Boehringer Mannheim, Indianapolis, IN)
Conclusions
DNA samples of up to 49 kb were concentrated
repeatedly without any loss of sample integrity.
Some loss of integrity was observed with a 125 kb
sample, although it was not complete and repre-
sents a small percentage of the total DNA in the
sample. For large fragments of DNA, centrifugal
ultrafiltration provides a fast and efficient method to
concentrate or desalt the sample. It results in high
recovery of intact product.
References
1. Takagi S, Kimura M, Katsuki M.
BioTechniques 1993;14(2):218–21.
2. PCR is covered by U.S. patents issued to
Hoffmann-LaRoche, Inc.
3. Sheng N, Zhang J, Whitton JL, McKee T.
BioTechniques 1993;14(5):781–4.
4. Ruano G, Pagliaro EM, Schwartz TR, Lamy K,
Messina D, Gaensslen RE, Lee HC.
BioTechniques 1992;13(2):266–74.
Figure 17.2 Specific plasmid DNA

and Bsu36 l digested BacPAK6 DNA (125 kb) was Lane 1: pBR322 starting material 1 2 3 4 5 6 7 8 9
Lane 2: pBR322 spun in Microcon
used. The samples were diluted and centrifuged as
30K NMWL device
described above. The retentates and starting Lane 3: pBR322 spun in Centricon
material were run on a 1% agarose gel in a 30K NMWL device
CHEF-DR® II pulsed field electrophoresis system
Lane 4: pSPT18 starting material
(Bio-Rad, Richmond, CA). Electrophoresis was Lane 5: pSPT18 spun in Microcon
performed at 200 V at 14 °C in 0.5X TBE with 30K NMWL device
ramped pulse from 1 to 6 seconds over 14 hours. Lane 6: pSPT18 spun in Centricon
The results with the lambda DNA mimic those 30K NMWL device
of the other samples run in these sets of experiments. Lane 7: pXT1 starting material
No adverse effects are noted after spinning the Lane 8: pXT1 spun in Microcon 30K NMWL device
Lane 9: pXT1 spun in Centricon 30K NMWL device
DNA at g forces up to 12,000 x g in the ultrafiltra-
tion units. However, the larger BacPAK®6 DNA does
show some degradation after ultrafiltration at both
5,000 and 12,000 x g (Figure 17.3, smearing in Figure 17.3 Large fragment DNA 1 2 3 4 5 6
lanes 5 and 6). Although a large percentage of the
Lane 1: Lambda DNA starting material
sample appears to be intact, there was loss of Lane 2: Lambda DNA spun in
integrity of the BacPAK6 DNA sample after the Microcon 30K NMWL device
concentration procedure. Lane 3: Lambda DNA spun in
Centricon 30K NMWL device
Lane 4: BacPAK DNA starting material
Lane 5: BacPAK DNA spun in
Microcon 30K NMWL device
Lane 6: BacPAK DNA spun in
Centricon 30K NMWL device

49
Protocols • Nucleic Acids
DNA Extraction from Agarose Gels with Montage
Gel Extraction Kit or Ultrafree-DA Centrifugal Filters
Introduction
The Ultrafree-DA device is designed to recover
100 to 10,000 bp DNA from agarose gel slices in
one 10-minute spin. It consists of a pre-assembled
sample filter cup with an agarose gel nebulizer and
a microcentrifuge vial. The device uses gel compres-
sion to extract DNA from the agarose. Centrifugal
force collapses the gel structure, drives the agarose
through a small orifice in the gel nebulizer and
captures the resultant gel slurry in the sample filter
cup. As the agarose is compressed at 5,000 x g,
DNA is extruded from the gel's pores. The gel
matrix is retained by the microporous membrane,
and the DNA passes freely through the membrane.
DNA can then be recovered in the filtrate vial.
The Montage Gel Extraction Kit consists of
50 Ultrafree-DA centrifugal filters as well as a
modified TAE buffer that allows the casting and
running of the gel from which the DNA fragment
is to be extracted.
DNA prepared with the Ultrafree-DA centrifugal
filter requires no further purification for most applica-
tions, including cloning and radioisotopic or
fluorescent DNA sequencing. Since agarose gel
electrophoresis has high resolving power, the small
and large non-specific amplification products that
frequently interfere with cloning and sequencing
after PCR (polymerase chain reaction) are com-
pletely removed from the product.
50
Materials
• Microcentrifuge
• Pre-assembled Ultrafree-DA centrifugal filter
device or Montage Gel Extraction Kit
• Modified TAE* electrophoresis buffer (40 mM
Tris-acetate, pH 8.0, 0.1 mM Na2EDTA)
• SeaKem agarose (FMC BioProducts) or
equivalent
• Long-wavelength UV lamp
• Scalpel or razor blade
Procedure
1. Electrophorese 30 µL of PCR product or other
DNA through a <1.25% ordinary agarose gel,
prepared in modified TAE buffer with ethidium
bromide (0.5 µg/mL).
2. Locate the band of interest with a long wave-
length UV lamp or transilluminator. With a razor
blade or scalpel, cut out the slice of agarose
(<100 µL or 100 mg) containing the band of
interest. Trim any excess agarose away from
band.
3. Place the gel slice into the gel nebulizer/sample
filter cup/filtrate vial assembly and seal the
device with the cap attached to vial.
*Modified TAE is recommended rather than TBE for the
following reasons: (1) TBE buffer strongly inhibits DNA
sequencing reactions while modified TAE buffer does
not. (2) Modified TAE has 0.1 mM Na2EDTA while
regular TAE has 1.0 mM Na2EDTA. The EDTA level at
0.1 mM Na2EDTA will not interfere with the magnesium
concentration in sequencing reactions and other
downstream enzymatic treatments, many of which are
dependent on magnesium.
4. Spin at 5,000 x g for 10 minutes.
Centrifugation forces the agarose through the Table 18.1 Effect of gel disruption on typical DNA recoveries from
gel nebulizer, converting it to a fine slurry that is agarose gels
captured by the sample filter cup. Extruded % DNA Recovered
DNA in electrophoresis buffer passes through % DNA Recovered from Gel Disrupted
DNA Size (bp) from Intact Gel by Gel Nebulizer
the microporous membrane in the sample filter
100 74 78
cup and collects in the filtrate vial.
400 39 ND
5. DNA in the filtrate is now ready for sequencing
or cloning without further purification. Discard 700 43 71
the gel nebulizer and sample filter cup and store 1000 55 77
the DNA in the capped filtrate vial. 2027 * 47
4361 14 35
Results 9416 * 32
Gel compression is a quick and easy technique for 23130 * 29
recovering DNA from an agarose gel slice.
* = Not detectable

51
Protocols • Nucleic Acids

RNA Purification with Microcon

or Centricon Centrifugal Filters

Introduction Methods
When working with RNA, introduction of RNase RNA Sample Integrity
contamination during sample preparation is of major The primary objective of the study was to determine
concern. Sample yield and integrity can impact the the effect of centrifugation on RNA integrity. The
efficiency of subsequent translations, hybridizations, second objective was to determine any gross
protein binding, antisense, or ribozyme studies. changes in the RNA that could be a result of contact
A series of experiments was performed to determine of the sample with the ultrafiltration devices
the effectiveness of using Amicon centrifugal ultra- themselves. An RNA transcript was made, using
filtration devices from Millipore to concentrate and MEGAscript T7 RNA polymerase (Ambion) and
diafilter RNA samples. The results indicate remarkably DNA template Riboprobe® Gemini (Promega)
high RNA recoveries and low adsorption losses— according to manufacturer’s protocols. This transcript
especially with prior membrane passivation. served as the starting RNA sample for the experi-
ments discussed below.
Figure 19.1 is an autoradiogram of labeled
RNA. It compares an untreated sample to samples
that were concentrated in Microcon 100K NMWL
and 30K NMWL devices, used directly as supplied.
The Microcon 100K NMWL unit was spun at a
force of 3,000 x g and the Microcon 30K NMWL
unit at 12,000 x g. As is evident from the auto-
radiogram, both unfiltered samples are virtually
indistinguishable from the starting material.
Similar results were obtained with Centricon units,
Figure 19.1 Concentration of RNA 1 2 3 spun at 1,000 x g (Centricon 100K NMWL unit)
transcript. RNA is intact and recovered or 5,000 x g (Centricon 30K NMWL unit).
1.4 kb
with high efficiency.
Lane 1: Starting material Diafiltration of RNA Samples
Lane 2: RNA concentrated in a Experiments were run to determine the RNA recovery
Microcon 100K NMWL device 0.68 kb
and diafiltration efficiency of Centricon devices.
Lane 3: RNA concentrated in a
Centricon 100K NMWL units were used to remove
Microcon 30K NMWL device
unincorporated, radiolabeled ribonucleotides from
the RNA transcript. Two mL of distilled water were
Table 19.1 TCA and total counts of unpurified and purified RNA samples placed into the Centricon sample reservoir, then
90 µL of the unpurified RNA transcript were added.
Unpurified Centricon-purified
Transcript Transcript The device was spun for 30 minutes at 1,000 x g,
TCA counts 70,000 68,500 then inverted and spun for 2 minutes at 1,000 x g
to collect the concentrated RNA (retentate). The
Total counts 136,500 69,000
retentate was diluted 1:10 with water. Total and
TCA precipitable counts were measured.

52
 If the Centricon device effectively removed the
unincorporated ribonucleotides, Total and TCA 100%
counts should be close in value. TCA counts of the
retentate sample and of the unpurified transcript 80%

RNA Recovered
(starting material) should also be similar if ultra-
60%
filtration resulted in high recovery of the transcript.
Average values (n = 3) of the Total and TCA counts
40%
appear in Table 19.1. For the retentate sample, the
Total and TCA counts are similar, indicating > 95% 20%
removal of the nucleotides. TCA counts of the samples
indicate that RNA recovery was also above 95%. 0%
0.025 0.25 0.5 1.0 5.0 10.0
RNA Concentration (µg/mL)
RNA Recovery
RNA recovery is a function of the initial RNA concen- Microcon Microcon Microcon Centricon
tration and the buffer salt concentration. Figure 19.2 Filtrate Membrane Retentate Retentate

shows RNA transcript recovery in Microcon 100K

NMWL and Centricon 100K NMWL units as a
Figure 19.2 RNA recovery in Centricon and Microcon concentrators. RNA
function of RNA concentration. For all concentrations
was concentrated as described in text. Cherenkov counts of Centricon retentate
evaluated, retentate recovery is above 85%, even and Microcon retentate, filtrate and membrane (n=3 units) were compared to total
at an initial concentration as low as 25 ng/mL. starting counts.
Figure 19.2 also displays the material monitored in
the filtrate and on the membranes of the Microcon
units. (Membranes were removed and counted 100%
without further treatment.) The amount of RNA in the
filtrate does not vary significantly as a function of 80%
concentration. The RNA recovered on the mem-
RNA Recovered

branes varies from <1% at high initial concentrations 60%

to 4% at the lowest concentration tested.

40%
Millipore’s Amicon centrifugal ultrafiltration
devices contain low-binding cellulosic Ultracel-YM
20%
membranes. Nitrocellulose is commonly used to
immobilize RNA, generally in high salt concentra- 0%
tions. Figure 19.3 shows RNA recovery as a 10 100 200 500
NaCl Concentration (mM)
function of buffer salt concentration. The data
show that both retentate and total recoveries fall as Filtrate Membrane Device Retentate
salt concentration increases. An increase in the
amount of material on the membrane is also seen
Figure 19.3 Effect of increasing buffer salt concentration on RNA recovery
(from 2% to 6%) as the salt concentration increases
with Centricon devices (two left bars at each concentration) or Microcon devices
from 10 to 500 mM. (two right bars at each concentration). Radiolabeled RNA samples were spun at
Similar trends are seen in the use of Microcon 1,000 x g for 30 minutes in a Centricon device or at 3,000 x g for 15 minutes
100K NMWL devices, except that retentate in a Microcon device. Retentate was collected by inverting the unit and spinning
recoveries fall to 60% at 500 mM salt. It was for 2 minutes (Centricon devices) or 1 minute (Microcon devices) at 1,000 x g.
Cherenkov counts of retentate, filtrate, membrane, and device (Microcon units
possible to count the devices themselves in addition
only) were compared to total starting counts.
to the filtrate and membrane. The results show a
slight increase in counts on the membrane with
increasing salt concentration. Significantly more
(from 9% to 17%) material remained on the device
as salt concentration increased.

53
 Passivation increased RNA recovery to 80%,
100% regardless of initial salt concentration (Figure 19.4).
This is attributed to a decrease in the amount of
80% RNA adhering to the device. When the devices
RNA Recovered

were passivated, RNA loss due to adsorption was

60%
reduced to 2%, regardless of salt concentration.
40%
Conclusion
20% The results shown here demonstrate that RNA
samples can be concentrated or diafiltered with
0%
Centricon and Microcon concentrators without loss
10 100 200 500
NaCl Concentration (mM) of RNA integrity. RNA recovery in the retentate is
typically > 85%. As the salt concentration of the
Filtrate Membrane Device Retentate buffer system increases, RNA retentate recovery
decreases, principally due to RNA adhering to the
device rather than to the membrane. RNA recovery
Figure 19.4 Improvement in RNA recoveries using Microcon devices (n= 6)
which were pre-treated with a 5% SDS solution. Samples were run as described in from solutions with high initial salt concentrations
Figure 19.3. can be significantly improved by pre-treating the
devices with a 5% SDS solution.

54
 Protocols • Nucleic Acids

Enzyme Removal with Micropure-EZ

Centrifugal Filters

Introduction Figure 20.1 Operation of Micropure-EZ device

The Micropure-EZ device is a convenient device
for removing enzymes from double-stranded (ds)
DNA ( 20 bp to >50,000 bp) solutions in a single
DNA Enzyme Salt
centrifugation. It can be used to remove enzymes Remove Enzyme
Micropure-EZ Restriction and other
whenever heat inactivation or phenol/chloroform enyzmes are absorbed
by Micropure-EZ.
extraction is impossible or impractical. After an dsDNA passes freely.
enzyme reaction, the modified DNA solution
Concentrate DNA
containing dilute protein is simply pipetted into a Remove Enzyme DNA is retained by
Micropure-EZ
Restriction and other
Micropure-EZ device that has been inserted into a enyzmes are absorbed Microcon
ultrafilter in Microcon.
Salts pass freely.
Assembly
vial (which is supplied) and centrifuged for 1 minute by Micropure-EZ.
dsDNA passes freely.
at 12,000 –14,000 x g. The DNA passes freely
Vial
into the vial (typically with 85– 90% DNA recovery) Recover
≥ 85% DNA
while the enzyme remains bound to the proprietary
membrane in the Micropure-EZ device. Removal
is defined as the complete absence of detectable
enzyme activity or, as in the isolated case of 30 seconds 3 minutes
T4 polynucleotide kinase, inconsequential residual
activity (<0.08%). For simultaneous concentration
of the enzyme-free DNA, place the Micropure-EZ
device into a Microcon microconcentrator (Figure
Method
20.1) before centrifuging again. The purified DNA
In order to ensure that each enzyme assay was
is suitable for cloning or for other enzymatic
detecting trace amounts of restriction enzymes in
manipulation.
Micropure-EZ filtrates, serial dilutions of Bgl I
While most enzymes were removed by the
(representing 2 units, 0.4 units, 0.08 units, and
Micropure-EZ device (Table 20.1, on page 57),
0.016 units of total activity in 10 µL) were carried
Millipore felt it was equally important to notify
out in a reaction mix* prepared with either sterile
customers of several enzymes that were not removed
DI water (sdH2O) or with sdH2O that had been
(Table 20.2). Millipore scientists were very careful to
filtered through Micropure-EZ and then incubated.
rule out enzyme inhibition as a mechanism. The
Comparison of the resultant restriction digests after
sensitivity of each assay to detect trace amounts
agarose gel electrophoreses verified that aqueous
of restriction enzymes in Micropure-EZ filtrates
extractables from Micropure-EZ devices did not
was determined by carrying out a dilution series
inhibit the enzyme. Also, these standard curves
(this is the method used by restriction enzyme
determined the sensitivity of the enzyme assay
manufacturers to estimate total activity).
(i.e., the theoretical amount of residual enzyme
activity that could be detected, if present).

*15.8 µL of sdH2O, 2 µL of 10X NEBuffer 2,

0.2 µL of 100X BSA, and 2 µL of Bgl I (10 units/µL)

55
 To stress test Micropure-EZ devices sufficiently
with enzymes and DNA, several extreme operating
conditions were used consistently. Micropure-EZ
devices were challenged with 50 units of Bgl I in
the presence of decoy DNA (1 µg of pBR322) and
5 µg of bovine serum albumin (BSA). Devices were
spun at 14,000 x g for 30 seconds. The filtrates
were then assayed for residual enzyme activity by
adding 1 µL of pUC19 DNA and 0.5 µL of 100X
BSA to the filtrate, mixing thoroughly and incubating
at 37 °C for 1 hour. After a brief centrifugation to
collect the condensate at the bottom of the micro-
centrifuge tubes, 1 µL of 0.5 M disodium EDTA
and 10 µL of 5X loading buffer were added to
stop the reaction.
The negative control was a portion of DNA
master mix. Control digests of pBR322 DNA and
pUC19 DNA were carried out separately.
Results and Discussion
The Bgl I standard curve made with the Micropure-
EZ-filtered sdH2O was indistinguishable from the
standard curve made with untreated sdH2O. The
plasmid DNA (1 µg of pUC19 and 1 µg of pBR322)
was cut to completion by 2 units of Bgl I after one
hour at 37 °C (Figure 20.2, lanes 1 and 6). None
of the enzymes shown in Tables 20.1 or 20.2 was
measurably inhibited by Micropure-EZ-filtered
sdH2O. The standard curves indicated that as little
as 0.08 units of Bgl I could be detected by this
assay (lanes 3 and 8).
The devices removed the 50 units of Bgl I as
evidenced by an absence of detectable activity in
the filtrates. In the lanes corresponding to the Bgl I
challenged Micropure-EZ inserts, some activity
was observed against the decoy pBR322 DNA,
as expected during its brief exposure to Bgl I
(particularly evident in lane 14). However, the
pUC19, which was added directly to the filtrate
and incubated, appeared completely intact
(lanes 11–14). Since the standard curve indicated
that 0.08 units would be detected if it were present,
we calculated that at least 99.8% of Bgl I was
removed and/or inactivated. Removal, rather than
inactivation, is the probable mechanism by which
Micropure-EZ devices operate, since in separate
experiments we were unable to detect any enzyme
(irrespective of activity) in Micropure-EZ filtrates
using very sensitive HPLC methods (data not shown).
Summary
Enzyme removal and excellent DNA recovery are
accomplished with Micropure-EZ devices in a single
60-second spin without any pre-wetting, binding,
washing or elution step. Cumbersome phenol/
chloroform extraction and the associated hazardous
waste accumulation are avoided. An additional line
of evidence for the suitability of Micropure-EZ
devices in molecular biology applications is that
restriction digest purified with Micropure-EZ devices
alone are readily ligated and cloned into plasmid
DNA (M.H.A.L., unpublished observations).
Micropure-EZ devices used alone or with Microcon
devices permit rapid and efficient sequential
enzymatic DNA manipulations.

Figure 20.2 Analytical agarose gel, demonstrating the detection limits for Bgl I and Bgl I removal by Micropure-EZ devices.
Lanes 1–4: Serial dilutions of BgI I representing 2 units, 0.4 units, 0.08 units, and 0.016 units of activity (respectively) against the DNA
master mix prepared in non-filtered sdH2O
Lane 5: Undigested DNA master mix used as substrate for the standard curve containing pUC19 and pBR322 DNA
Lanes 6–9: Inhibition control: serial dilutions of BgI I representing 2 units, 0.,4 units, 0.08 units, and 0.016 units of activity (respectively)
against the DNA master mix prepared in Micropure-EZ-filtered sdH2O
Lane 10: Molecular weight standard: 1 kb DNA ladder (Gibco-BRL, Gaithersburg, MD)
Lanes 11–14: Filtrates incubated with
pUC19 DNA after challenging
Micropure-EZ devices with
50 units of BgI I, BSA and
decoy pBR322 DNA
Lane 15: Uncut pBR322
Lane 16: BgI I–cut pBR322
Lane 17: Uncut pUC19
Lane 18: BgI I–cut pUC19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

56
 Table 20.1 Enzymes removed by Micropure-EZ devices

In tests, all enzymes were removed from solutions containing DNA or RNA (in the case of RNase A). Five µg of bovine serum
albumin were also present during removal of restriction enzymes. Removal was indicated by undetectable or inconsequential
enzyme activity in filtrate (< 0.08% residual in the case of T4 polynucleotide kinase).
Enzyme Challenge Enzyme Challenge Enzyme Challenge
AMV reverse transcriptase 50 U Apa I 100 U Mlu I 50 U
Calf intestinal alkaline 10 U BamH I 100 U Nco I 50 U
phosphatase Bcl I 50 U Nde I 100 U
DNase I (bovine pancreas) 10 U Bgl I 50 U NgoM I 50 U
Exonuclease III (E. coli) 100 U BsiW I 70 U Nhe I 25 U
MMLV reverse transcriptase 600 U BssH II 20 U Not I 50 U
Mung bean nuclease 50 U BstN I 50 U Nru I 50 U
Proteinase K (Amresco) 5 µg Dpn I 100 U Pst I 100 U
T4 DNA ligase 2,000 U* EcoR I 100 U Sac I 100 U
T4 DNA polymerase 15 U Hae III 100 U Sac II 100 U
T4 polynucleotide kinase** 50 U Hinc II 50 U Sal I 100 U
Taq DNA polymerase 5U Hind III 100 U Sca I 50 U
Terminal deoxynucleotidyl 45 U Hpa I 25 U Sph I 25 U
transferase Kpn I 50 U Sst I 50 U
Acc I 50 U Mbo I 25 U Xho I 100 U
*New England Biolabs unit definition
**T4 polynucelotide kinase is not recommended with oligonucleotides (dsDNA only). The results suggest that this kinase mediates the binding of
oligos to the membrane in Micropure-EZ device, causing oligo loss.

Table 20.2 Enzymes not removed by Micropure-EZ devices

In tests, Micropure-EZ did not remove the indicated number of units of the listed enzymes. It may be effective in removing a lower
number of units.
Enzyme Challenge Enzyme Challenge Enzyme Challenge
Bacterial alkaline phosphatase 0.6 U ApaL I 10 U Msp I 100 U
DNA polymerase I (Klenow) 20 U Bgl II 50 U Pvu I 25 U
Exonuclease I 50 U BsoB I 50 U Pvu II 50 U
Pfu DNA polymerase 2.5 U Cla I 25 U Sau3A I 20 U
Vent™ DNA polymerase 4U Eae I 15 U Sfi I 50 U
Shrimp alkaline phosphatase 1U EcoR V 50 U Sma I 25 U
RNase A (bovine pancreas) 1 µg Hinf I 50 U Xba I 50 U
T4 RNA ligase is not recommended. Results suggest this ligase mediates binding of nucleic acids to the membrane in Micropure-EZ causing sample loss.

57
Protocols • High Throughput
High Throughput Applications
Figure 1. MultiScreen
Filter Plate with Ultracel-10
ultrafiltration membrane
The MultiScreen filter plate with Ultracel-10
membrane provides a new method for high
throughput sample prep. The ultrafiltration-based
filter plate is designed for use with centrifugation
and is compatible with standard microtiter plates,
instrumentation, and liquid handling equipment.
All of the publications summarized below
can be supplied by your local Millipore office or
downloaded from www.millipore.com/ultracel.
58
Nucleic Acid Purification
and Concentration
AN1040EN00
The MultiScreen Filter Plate with Ultracel-10
membrane can be used to purify and concentrate
single-stranded oligonucleotides and double-
stranded DNA fragments, as well as plasmid and
genomic DNA.
Concentration of Proteins
in Cell Lysate
AN1424EN00
The MultiScreen filter plate with Ultracel-10
membrane can be used to concentrate whole cell
lysates without loss of protein and with high
reproducibility across the plate. Applications include
parallel protein purification, protein concentration
and buffer exchange in cell lysates for subsequent
separation or assaying.
Purification of Serum Peptides
for Biomarkers Research
AN2010EN00
This study is a high throughput version of the
application nate on page 24 of the Protocols
section of this handbook.
Enzymatic Activity Recovery
AN2011EN00
This study demonstrates the use of the MultiScreen
plate with Ultracel-10 membrane for concentrating
alkaline phosphatase without loss of biological
activity and with high reproducibility across
the plate.
Glossary
Asymmetric Membrane1
Membrane constituted of two or more struc-
tural planes of non-identical morphologies.
Batch Process
A fixed volume of solution contained in a
tank to which the concentrate is returned
during the process.
Composite Membrane1
Membrane having chemically or structurally
distinct layers.
Concentration Polarization
Accumulation of rejected solute on the
membrane surface. Depends on interactions
of pressure, viscosity, crossflow (tangential)
velocity, fluid flow conditions, flow channel
conditions and temperatures.
Crossflow (Tangential Flow)
Solution flows across (tangential to) a mem-
brane surface. Facilitates back diffusion of
solute from that surface into the bulk solution,
counteracting concentration polarization.
Diafiltration
Removal of small components from the
retained species during ultrafiltration by
adding water or buffer solution to the
retentate. See page 7 for further discussion.
Dialysis
Diffusive transport of ions or other small
molecules through a membrane barrier
that contacts solvent on both sides.
Downstream1
Side of a membrane from which the
permeate emerges.
Feed (Sample)
The starting solution (sometimes the solution
remaining upstream of the membrane).
Fluid Velocity
The flow rate of solution across the mem-
brane surface in cross (tangential) flow.
Related to hydraulic pressure drop.
Flux
The filtration rate through the membrane per
unit area.
Fouling
Irreversible decline in membrane flux due to
deposition and accumulation of submicron
particles and solutes on the membrane
surface. Also, crystallization and precipita-
tion of small solutes on the surface and in the
pores of the membrane. Not to be confused
with concentration polarization.
Membrane1
Structure having lateral dimensions much
greater than its thickness, through which
mass transfer may occur under a variety of
driving forces.
Molecular Weight Cut-off (MWCO)
See Nominal Molecular Weight Limit.
Nanofiltration1
Pressure-driven membrane-based separation
process in which particles and dissolved
molecules smaller than about 2 nm are
rejected.
Nucleotide Cut-off (NCO)
The number of nucleotides in a DNA
fragment (single- or double-stranded) at
which 90% of the fragment is retained by
the membrane.
Nominal Molecular Weight Limit
(NMWL)
The molecular weight at which at least 90%
of a globular solute of that MW is retained
by the membrane.
Permeate (Filtrate, Ultrafiltrate)
The solution passing through the membrane,
containing solvent and solutes not retained
by the membrane.
Plugging
Accumulation of debris on the fluid flow path,
restricting or blocking flow.
Rejection
The fraction of solute held back by the
membrane. Can be measured at any point in
the process or averaged over the run.
Retentate (Reject Stream, Concentrate)
The solution containing the retained (rejected)
species.
Retention Factor1 (rF)
Parameter defined as one minus the ratio
of permeate concentration to the retentate
concentration of a component. Note:
r F = 1 – [p]/[r] where [p] = concentration
of solute in permeate, and [r] = concentra-
tion of solute in retentate.
Reverse Osmosis1
Liquid-phase pressure-driven separation
process in which applied transmembrane
pressure causes selective movement of solvent
against its osmotic pressure difference.
Tangential Flow Filtration (TFF)
Flow through a membrane device in which
the fluid on the upstream side moves parallel
to the membrane surface.
Transmembrane Pressure (TMP)
The driving force in ultrafiltration. In a stirred
cell, equivalent to gas pressure. In centrifugal
devices, it is related to g-force. In a flowing
system, TMP decreases as the stream moves
from inlet to outlet. Average TMP =
[(Pin + Pout)/2] – Ppermeate
Ultrafiltration1
Pressure-driven membrane-based separation
process in which particles and dissolved
molecules smaller than 0.1 µm and larger
than about 2 nm are rejected.
Yield
Amount of species recovered at the end of
the process as a percentage of the amount
present in the feed solution.
References
1. Terminology for membranes and
membrane processes (IUPAC
Recommendations 1996). IUPAC,
Journal of Membrane Science.
1996;120(1):149–59.
59
Acknowledgements
Millipore wishes to acknowledge the efforts of the following employees in developing
this reference book:

Elena Chernokalskaya, Ph.D.

Manager, Proteomics Applications

Mark Kavonian
Worldwide Group Product Manager
Protein Research Products

Richard Leary
Technical Service Specialist

Jack T. Leonard, Ph.D.

R&D Director, Life Science Research Applications

Peter Rapiejko

Millipore also wishes to acknowledge the following individuals for their contributions:
Eduardo Vottero, University of British Columbia
Peter A. Lemaire and Dr. James Cole, University of Connecticut
Gary Smejkal, Proteome Systems, Woburn, MA
Leonid Kryazhev, Genome Quebec, Montreal, Canada
Mathew L. Thakur, Thomas Jefferson University Hospital, Philadelphia, PA
Mark Merchant, Helena Laboratories, Beaumont, TX
Department of Forensic Biology, New York City
Patrick O. Brown, Max Diehn, Ash Alizadeh, Stanford University School of Medicine
Dr. Sophia N. Karagiannis, GKT School of Biomedical Sciences, London

60
Patent
PCR is covered by US patents issued to Hoffmann-LaRoche, Inc.

Trademarks
Millipore, Amicon, Biomax, Centricon, Centrilutor, Centriplus, Centriprep, Durapore, Immobilon,
Microcon, Micropure, Milli-Q, Minicon, Montage, MultiScreen, Pellicon, Prep/Scale, PROSEP, Ultracel,
Ultrafree, Zip, and ZipTip are trademarks of Millipore Corporation.
ProteoPrep is a trademark of Sigma-Aldrich Biotechnology LP.
ProteomIQ, ElectrophoretIQ, IsoelectrIQ, and GelChips are trademarks of Proteome Systems.
SpectraFLUOR is a trademark of Tecan Group AG.
DryStrips, Sephadex, Sephacryl, Sepharose, and Storm are trademarks of Amersham Biosciences AB.
CHEF-DR II and Chelex are trademarks of Bio-Rad Laboratories.
MEGAscript is a trademark of Ambion Inc.
GenePrint, Flexi and Riboprobe are trademarks of Promega Corporation.
SeaKem is a trademark of FMC.
AmpFLSTR and Voyager-DE are trademarks of Applera Corporation or a subsidiary.
Triton is a trademark of Union Carbide Chemicals & Plastics Technology Corporation.
Biofuge is a trademark of Kendro Laboratory Products, Gmbh.
NuPage, SimplyBlue, Superscript and Xcell Surelock are trademarks of Invitrogen Corporation.
Qiagen is a trademark of Qiagen Gmbh.
Speed Vac is a trademark of Thermo Savant.
BacPAK and Talon are trademarks of Becton, Dickinson and Company.
Vent is a trademark of New England Biolabs, Inc.
Coomassie is a trademark of Imperial Industries, PLC.
Tween is a trademark of Atlas Powder Company.
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