Protein Purification Tutorial

Protein purification tutorial

TITLE PAGE
graphic

"Proteins" a word derived from the Greek word, Proteios,

which means "of the first rank" was coined by Jons J.
Berzelius in 1938 to stress the importance of this group of
organic compounds. Proteins have been described as the
servants of life. Most proteins are enzymes but in addition
there are also regulatory and structural proteins.
Protein Purification Tutorial Intro –Why purify proteins?

• During the last decade, the genomic sequence of many organisms has been determined. The wealth of
information in these genomes has dramatically changed research. If you can identify the gene within the
genomic DNA sequence, you already know the amino acid sequence of the encoded protein and the
regulatory elements that control the expression of this gene. You don’t, however, know the structural
organization of the protein or how it is regulated. If you do not have a genomic database or cannot identify
the protein coding region within the genome, you may want to purify the protein in order to help you to
isolate the gene.
– 1) If you have not yet isolated the gene that encodes the protein, you may want to purify the protein for
any of the following reasons:
– The purified protein can be used to determine the amino acid sequence. The sequence can then be
used as a probe to help in the isolation of the gene.
– The purified protein can be used to produce antibodies that can be used as a probe to help in the
isolation of the gene.
– The purified protein can be analyzed by mass spectroscopy. The molecular weight can then be used
as a screen of the genomic sequence for the gene.
– 2) If you have already isolated the gene that encodes the protein, you may want to purify the protein
for any of the following reasons:
– Pure proteins are required for structural analysis.(x-ray crystallography and NMR spectroscopy)(Figure
1).
– Pure proteins are required to study enzyme function.
– Pure proteins are needed in order to learn about what other proteins or nucleic acids they interacts
with. (Figure 2)
– Pure proteins are needed for studies of enzyme regulation (are their regulatory subunits, is it regulated
by phosphorylation, is the protein regulated by interaction with other proteins.)
Protein Purification Tutorial Objectives

• In this tutorial you will:

– 1
– 2
– 3
– 4
– 5
Protein Purification Tutorial Intro – table of common techniques

Table of common methods of protein purification

• Purification procedures attempt to
Property Methods maintain the protein in native form.
Although some proteins can be
Precipitation with
solubility
ammonium sulfate
re-natured, most cannot!
(salting out)* • To purify a protein from a mixture,
Size / shape Size-exclusion biochemists exploit the ways that
chromotography individual proteins differ from one
another. They differ in:
Isoelectricpoint Ion exhange
(charge) chromatography – Thermal stability*

binding to small Affinity

molecules chromatography

*Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
It is commonly one of the first steps in a purification scheme.
*For most protein purifications, all steps
are carried out at ~5°C to slow down
degradative processes.
Protein Purification Tutorial Intro side bar

Picture of protein gel with lanes

showing sequential purification
steps

Purification Table

Procedure Fraction vol Total Prot Activity Specific activity Purificati

Yield
(ml) (mg) (units) Units/mg on factor
Crude cellular 1400 10000 100000 10 1 100%
extract
Size-exclusion 90 400 80000 200 20 80%

Ion exchange 80 100 60,000 600 3 75%

Add row with ammonium sulfate data. Include two

colums at end called Purification factor and Yield.
Note: The type and order of steps are customized for each
protein to be purified. An effective purification step results in
a high yield (minimal loss of enzyme activity) and large
purification factor (large increase in specific activity).
 Intro – flow chart. Purification is a multi step
Protein Purification Tutorial procedure

Purification is a multi-step procedure.

Sample
Separation
technique

Repeat with another

Fractionation
separation
technique until pure

No
Assay total protein
Assay enzyme activity

No yes Combine Pure?

Set aside Is there activity? Monitor purity
Fractions

yes

Prepare for analytical technique

Protein Purification Tutorial Intro – first steps.

• First steps
– 1. Source. A good source is
cheap and readily available.
Many proteins are enriched in
specific tissues, for example
hemoglobin in blood. For this
reason, these tissues may be
excellent sources for your protein.
– 2. Assay: Most assays are
chemical reactions catalyzed by
specific enzymatic activities.
Proteins that have no activity are
usually assayed using SDS
polyacrylamide gels.
Protein Purification Tutorial Assay – develop an assay

• First steps – Develop an Assay

3. An assay for an enzyme is a

method for quantifying its activity.

Since the assay is repeated many

times, it is important that it be a
simple procedure. Usually
enzyme activity is monitored as a
change in absorbance which can
be measured using a
spectrophotometer. For example
an assay for ribonuclease
measures the change in
absorbance that accompanies the
breakdown of RNA to
ribonucleotides.
Protein Purification Tutorial Intro – Preparing the sample (Crude Extract)

First steps: Preparing the sample – Crude extract.

Protein from cells or tissue

Supernatant with
Soluble protein
Break cells,
tissue, or organ

Blender,
homogenizer,
sonication,
pressure,
Microbial cells psmotic Pellet with intact cells, organelles, mem
or tissue
Protein Purification Tutorial Segway to Chromotography

• Some kind of graphic. • Note: In order to isolate sufficient

quantities of protein, you may
need to start with kilogram
quantities of source tissues.
These amounts can best be
handled using precipitation
methods (e.g. ammonium sulfate
precipitation). Later in the
purification, large columns can be
used to handle gram to milligram
quantities. Amounts handled on
gels are typically in microgram
quantities.
 Intro – flow chart. Purification is a multi step
Protein Purification Tutorial procedure

Purification is a multi-step procedure.

Sample
Separation
technique

Repeat with another

Fractionation
separation
technique until pure

No
Assay total protein
Assay enzyme activity

No yes Combine Pure?

Set aside Is there activity? Monitor purity
Fractions

yes

Prepare for analytical technique

Protein Purification Tutorial Over view of the apparatus

Column Chromotography –general case

– Glass column
– Reservoir
– Solid matrix – beads
– Solution of protein.
– Effluent.
– Other terms?

We think it would be better to have a resevoir

attached to a cap on the top of the column via a
plastic tube, show minimal liquid on top of the
column bed, show a tube coming out of the
bottom of the column that has a clamp of
stopcock. Perhaps a schematic on the left and a
photo of an actual column running in a cold box
over a fraction collector on the right.
Should emphasize that the basic set=up is the
same for all column types, but the
characteristics of the beads vary.
Protein Purification Tutorial Load sample (4 protein mix)

• Image of apparatus with protein • TEXT. The crude extract is

mixture layered on top placed on top of the solid matrix.
(In this case we are using a
mixture of 4 proteins, indicated by
different colors.)

• (As the animation proceeds)

The proteins move at different rates
through the matrix based on the
properties of the proteins and the
type of column beads.
Note - this should be shown as a
single band (possibly brown or
striped with four colors)
Protein Purification Tutorial Collect fractions.

• Fractionation • Text:
As the column separates the proteins
in the mixture, the “effluent” drips
into a series of fraction tubes that
are moving at a specific rate of
speed. These tubes are called
fractions.

Here we are showing 20 tubes.

Fraction collectors in most labs
have about 75-200 tubes.

Be sure to remove color from

column as it drips into the tubes
below! If the sample is spread over
three tubes, the center tube will be
darker in color.
Protein Purification Tutorial --3 questions—

• In reality, proteins aren’t color- 1. How do we know which

coded so we must ask ourselves fractions contain protein?
these 3 questions:
3. Which of those fractions
contain the desired protein?

5. How do we assess the purity?

???
Protein Purification Tutorial Question 1 – take A280 Screen 1.

Question 1. How do we know which fractions contain protein?

• Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
Aromatic amino acids absorb light
at this wavelength causing all
proteinsTake
to have
A280 absorbance at
Fraction
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 280nm. Many fraction collectors
measure the A280 as the column
is running.
Protein Purification Tutorial Question 1 – take A280 Screen 2 .

Question 1. How do we know which fractions contain protein?

• Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
Aromatic amino acids absorb light
at this wavelength causing all
Fraction proteins to have absorbance at
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
# 280nm. Many fraction collectors
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 measure the A280 as the column
A280 is running.
• The A280values
Plot valuescan be plotted
against the fraction number in is
what is called an elution profile.
Protein Purification Tutorial Question 1 – take A280 Screen 3 .

Question 1. How do we know which fractions contain protein?

• Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
• The values can be plotted against
the fraction number in is what is
Fraction called an elution profile.
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280 • Notice the peaks on the graph.
These indicate where the fractions
Peaks are that contain protein.

A280

Fraction #
Protein Purification Tutorial Question 1 – take A280 Screen 4 .

Question 1. How do we know which fractions contain protein?

• Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
• The values can be plotted against
the fraction number in is what is
Fraction called an elution profile.
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280 • Notice the peaks on the graph.
These indicate where the fractions
Peaks are that contain protein.

A280

Fraction #
Protein Purification Tutorial Question 2 – take A280 Screen 1 .

Question 2. Which fractions contained the desired protein?

• Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.

Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
# Fraction
#
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280
Enz. Assay.

A280

Fraction #
 Protein Purification Tutorial Question 2 – take A280 Screen 2.

Question 2. Which fractions contained the desired enzyme?

• Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 • Notice the results of the enzyme

Fraction
#
assay. The highest activity
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280 corresponds to one of the peaks.
EnzAssay
Results
Need to substitute values for the colored
spots since we are switching to an absorbance Now we can have them discard
based assay. tubes that don’t have enzyme
activity.

A280

Fraction #
 Protein Purification Tutorial Question 2 – take A280 Screen 3.

Question 2. Which fractions contained the desired protein?

• Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 • Notice the results of the enzyme

Fraction
#
assay. Notice that the highest
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280 activity corresponds to one of the
peaks.
EnzAssay
Results

A280 • Discard the fractions that don’t

contain protein by clicking on the
tubes that don’t contain protein.

Fraction #
Protein Purification Tutorial POOL fractions – screen 1

Combine (pool) the fractions with activity.

1. NEXT We want to pool the

fractions that have enzyme
activity.

Fraction
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

It may be useful to consider more than just the activity of a

fraction. Specific activity is a measure of the amount of
enzyme activity per amount of protein (units/mg). The
higher the specific activity, the higher the purity. When
pooling fractions, judgement is needed as to whether to
optimize yield or specific activity. Pool
fractions

Fraction #
Protein Purification Tutorial POOL fractions – screen 2

Combine (pool) the fractions with protein and activity.

1. The fractions are pooled together.

How do we monitor the progress of

the purification?

Fraction #
Protein Purification Tutorial Assess purity – screen 1

– Look at it on a gel. Even when

Question 3: Is this pooled sample pure? How do you monitor the progress?
overloaded, if only one band is
visible, it is likely to be pure and a
Standards | Crude Ext. | Pooled fractions
monomer, a homo-dimer, or a
homo-multimer. If not a single
band, additional bands that purify
in parallel, will remain proportional
throughout the purification. If a
Purification Table band co-purifies, it is likely to be a
subunit for the enzyme.
Purification Table
Procedure Fraction vol
–TotalCalculate
Prot the specific activity
Activity by
Specific activity
(ml) (mg)doing a careful
(units) quantitative
Units/mgassay
Crude cellular 1400 10000for enzyme100000activity/total10protein.
extract
Separation 90 400 80000 200
method 1

Results:
2. Gel shows more than one band.

Since the sample is not pure, you must pass pooled sample through another separation technique

Another separation
Protein Purification Tutorial Assess purity – screen 2

Question 3: Is this pooled sample pure? How do you monitor the progress?

Standards | Crude Ext. | Pooled fractions – Look at it on a gel. A monomer

should have one band.
– Calculate the specific activity by
doing a careful quantitative assay
for enzyme activity/total protein.
Purification Table Purification Table
Procedure Fraction vol Total Prot Activity Specific activity
(ml) (mg) (units) Units/mg

Crude cellular 1400 10000 100000 10

extract
Separation 90 400 80000 200
method 1
Separation 8 4 60000 15000
method 2

Results:
2. Gel shows one band
3. Specific activity is 15000. Looks good

Your protein seems pure. YOU’RE DONE!!!.

Protein Purification Tutorial Intro – table of common techniques

Table of common methods of protein purification

Property Methods

solubility Precipitation with

ammonium sulfate
(salting out)*
The 3 separation techniques of chromotography
size Size-exclusion
chromotography

Isoelectricpoint Ion exhange

(charge) chromatography

binding to small Affinity

molecules chromatography

Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
It is commonly one of the first steps in a purification scheme.
Protein Purification Tutorial Gel filtration 1 -basis

Here’s our sample mix of proteins.

Our goal is to purify protein #….
• Gel filtration column
chromotography separates
proteins on the basis of size.

• We will start with 4 proteins.

• You will want to purify the “yellow
60 Kd 20 Kd 20 Kd 5 Kd one”
Low pI (6) Low pI (7) Medium pI (7) Hi pI (8)
Protein Purification Tutorial
Protein Purification Tutorial Gel filtration 2 - close up of beads

• The matrix of a size-exclusion

chromatography column is porous
beads.

Run column

Need two pore sizes (other size

bigger than black proteins, smaller
than existing pores.)
Protein Purification Tutorial Gel filtration 3 - run close up of column

• The matrix of a gel filtration

column are beads with pores.

• The large green proteins can’t fit in

pores so flows faster.
• The red/yellow medium sized
proteins get trapped in the pores.
• The black small proteins stay
trapped in pores longer.

We’re wondering how this will work in

animation. The black can permeate all
pores and the space between beads, the
yellow and red can permeate the space
between beads and larger pores, the
green will be restricted to the space
between beads.
Protein Purification Tutorial Gel filtration 4 - zoom out

Click on the peak that represents the

protein of the largest molecular
weight?

Be sure to keep in mind that the colors will either

be in the column or in the tubes, not both!

Tubes march in from left A280

Fraction #
Protein Purification Tutorial Gel Filtration 5.

• • Many columns are commercially

made. Here are some examples.
This could be moved to the earlier view of
the porous beads.

Fig 1.1. Scanning electron micrograph

of an agarose gel. Magnification x 50,000.
Ref. Anders S. Medin,PhD Thesis,
Uppsala University 1995.

Sephadex./
Protein Purification Tutorial ION –EXCHANGE 1

• Ion-exchange column
chromotography separates
proteins on the basis of charge.

• We will start with 4 proteins.

• pH 7.2
60 Kd 20 Kd 20 Kd 5 Kd • pos charged column
Low pI (6) Low pI (7) Medium pI (7) Hi pI (8)

Need to include a slide on how to determine the charge on a protein,

given its pI and the pH.
Protein Purification Tutorial Ion Exchange 2 – loaded proteins

• The matrix of an ion exchange is

positively charged.
• What do you think will happen?
pos

pos

Run column
pos

pos

pos

pos
Protein Purification Tutorial Ion Exchange 3 –column run

• The matrix of an ion exchange is

positively charged.
pos
• Only the pos charged proteins run
through the pos charged column.
pos The others “stick” to the column.

pos
These beads are porous, too, so you can show the
proteins moving right through the beads in the
animation, ifyou like.
pos

pos

pos
Protein Purification Tutorial Ion Exchange 4- zoom out

Only the POS charged proteins run

through the column.

How can we elute the other proteins?

20 1

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 5 - zoom out

Increase the salt. Add salt

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 6 – after salt

Increase the salt. Add salt

What protein will come of the column
next?
- - ---

Don’t show the charges on the color spots -

students should figure this out on their own!

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 7 – after salt

Increase the salt.

What protein(s) will come of the
column next? + --- ---
Feedback statement.

Run column

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 8 – after salt

Red and yellow will have the same

neg charge and will co-elute.

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 9 – Increase salt conc. Again.

Add salt
Increase the salt concentration

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 10 – rum column prompt.

Run the column.

Run column

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 11 – results.

Run the column.

Run column

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 12 – results.

Notice that 2 of the proteins eluted at

the same time. Why?
Is our protein pure? We were
supposed to purify the red one

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Affinity Chromotography.

• Affinity Chromotography • See notes below

Protein Purification Tutorial Monitoring progress.

• Monitoring progress. • We need some info on the SDS

page and the specific activity.
• See Jim’s hand written notes as a
guide.

Place final table here - we can include the thing plot with protein conc going
down, enzyme amount remaining constant, and specific activity on the rise.