Techniques in protein separation,

purifications and determination

.

1
Object : to separate a particular protein from all other
proteins and cell components 2
Introduction of proteins

Transcriptio
n
Translation

Reverse mRNA Protein

Transcriptio
DNA n
Replication
3
Protein, An introduction!!

4
 Protein functions
Enzymes: substrate binding, side chain in catalysis
Signaling: cell membrane
Transport and storage: hemoglobin transports oxygen
Structure and movement: collagen, keratin, actin and
myosin for muscle contraction
Nutrition: casein and ovalbumin
Immunity: antibodies
Regulation: transcription factors

5
 Protein analysis
1. Purification: to obtain enough pure sample for study

2. Sequencing: determine the primary structure of a pure

protein sample
3. Mass determination: determine the molecular weight
(MW) of an interested protein.

4. X-ray crystallography and NMR: determine the tertiary

structure of a given sample.

6
Techniques for proteins (enzymes) purification
Cell lysis techniques - osmolysis, mechanical disruption-high
speed blender, homogenizer, French press, sonication
Salting out and salting in
Chromatography
Ion exchange
Size exclusion
Affinity
others
Dialysis
Electrophoresis
SDS PAGE
Isoelectric focusing
7
 Protein purification

An essential experimental step prior to

study of any individual protein

To purify the interested protein from

other proteins and nonprotein
molecules existing in the cells

8
 Why purify a protein?

Characterize function, activity, structure

Use in assays
Raise antibodies
many other reasons ...

9
Main Steps in Protein Analysis
1. Extraction of proteins.
2. Purification of the protein.
3. Structural Characterization of the protein.

10
 1. Protein Extraction
Involves the efficient extraction of the protein in a
biologically active form from tissue.

During this process, inactivation of proteolytic enzymes is

required.
Why ? Not to cause degradation of the proteins contained
in the tissue.

Extraction of protein: homogenize it in a physiological

buffer (0.05M NaPO4, PH 7.4) containing proteolytic enzyme
inhibitors (EDTA)

11
I.1 Protein Isolation
Selection of Starting Material

Sources
animal or plant tissues
microorganisms

12
13
 2. Protein Purification
Proteins can be purified according to certain
properties they possess.
These properties allow us to employ different
techniques in purifying proteins.
Protein Property Technique

Solubility Differential Precipitation

Molecular Size Gel Filtration Chromatography
Molecular Charge Ion Exchange Chromatography
Hydrophobicity Reversed Phase HPLC
Biological Activity Affinity Chromatography

14
 Differential Precipitation
Precipitation: the process of formation of a solid
that was previously held in solution.
The solid is separated from the solution.

When NH4 SO4 or polyethylene glycol are added

to a protein solution, a precipitate forms and it can
be separated from the solution after centrifugation.
If the concentrations of NH4 SO4 or polyethylene glycol are
increased, more precipitate forms.

15
Based on Solubility
Change in pH (Isoelectric precipitation ) :
A procedure in which the pH of the protein
mixture is adjusted to the pI of the protein to be
isolated to selectively minimize its solubility.

solubility
pI pH
16
 Change in ionic strength (Differential
precipitation)
Salting in
- At low ionic strength, increases in the
concentration of dissolved ions leads to an
increase in solubility by weakening the interaction
between individual protein molecules.
Interactions between protein molecules leads to
aggregation (i.e. insolubility of proteins)

17
Salting out
As the ionic strength increases, they out
compete the proteins for water molecules
and the proteins become less soluble,
aggregate, and fall out of solution.

18
Concentration of extracts
Freeze drying
Dialysis
PEG precipitation
Concentration / fractionation by salting out
Ammonium sulphate precipitation
Ultraflitration
Desalting
Size fractionation
 Concentration of salts Salting out
Addition of the right amount
of (NH4)2 SO4 can selectively
precipitate some proteins but
not others. because it
competes for the solvent
(H2O) needed to solvate the
macromolecules.
So high [salt] removes the
solvation sphere from the
protein molecules and they
come out of solution
Protein Precipitation using
ammonium sulfate.

20
 Salting out
Mixture of proteins in buffer:
A: precipitates at salt [ ] = 15%
B: precipitates at salt [ ] = 25 %
C: precipitates at salt [ ] = 35%

1) Add (slowly) (NH4)2SO4 to

20%
Pellet: protein A 2) Centrifuge to pellet
precipitated proteins

Supernatant: protein B + C

1) Add (slowly) (NH4)2SO4 to

30%
Pellet: protein B 2) Centrifuge to pellet
precipitated proteins
Supernatant: protein C

21
Based on Molecular Size

Centrifugation
Process of subjecting a suspension of sample
at greatly increased gravitational field
(centrifugal force) by rapidly rotating a
receptacle containing the sample which will
lead to sedimentation of particles.
Application:
Differential Centrifugation
For separation of crude mixtures
of cellular components
22
23
 Dialysis
Dialysis-a process that separates molecules
according to size through the use of semipermeable
membranes containing pores of less than
macromolecular dimensions.

Pores in the membrane allow solvents, salts and

small metabolites to diffuse across but block larger
molecules.
Cellophane (cellulose acetate) most commonly used
dialysis material.
Usually used to change the solvent in which the
protein is dissolved in.
Can also be used to concentrate a protein solution by
placement in a polymeric dessicant (PEG) which
cannot go through the membrane but absorbs water
through the membrane. 24
Dialysis
It is the movement
of molecules by
diffusion from high
concentration to
low concentration.

A process that
separates molecules
by the use of a semi-
permeable membrane.

25
Size-dependent diffusion Gel filtration

Start

End

Dialysis 26
 Concentration - Ultrafiltration
When macromolecular solution is forced under
pressure thr. a semi-permeable bag/disc.

Protein solution
N2 pressure

Excess aqueous phase

Ultrafiltration
membrane
The principal properties of proteins
used for purification

1. Size: gel filtration chromatography

2. Charge: ion-exchange chromatography,

isoelectric focusing, electrophoresis
3. Hydrophobicity: hydrophobic interaction
chromatography

4. Affinity: affinity chromatography

28
Column Chromatography
Basis of Chromatography
Different compounds distribute themselves to
a varying extent between different phases
Interact/distribute themselves
In different phases
2 phases:
Stationary: samples interacts with this phase
Mobile: Flows over the stationary phase and
carries along with it the sample to be separated

29
Column Chromatography

30
 Types of liquid chromatography

Adsorption Chromatography
Proteins bind to stationary phase
Proteins eluted by altering mobile phase
Includes: affinity, hydrophobic interaction, ion exchange,
and chromatofocusing

Solution Phase Chromatography

Proteins do not bind to stationary phase
Progress of proteins through column impeded by matrix of
stationary phase
Includes: size exclusion chromatography (aka gel filtration)

31
 Size-Exclusion/Gel-Filtration
Separates molecules based on size.

Stationary phase composed of cross-linked gel particles.

Extent of cross-linking can be controlled to determine
pore size
Smaller molecules enter the pores and are delayed in
elution time. Larger molecules do not enter and elute
from column before smaller ones.

32
 Gel filtration chromatography
Total bed volume of the column
Vt = Vx + V0

Vx = volume occupied by gel beads

V0 = volume of solvent space surrounding gel; Typically 35%

Elution volume (Ve) is the volume of a solvent required to elute a given

solute from the column after it has first contacted the gel.

Relative elution volume (Ve/V0) is the behavior of a particular solute

on a given gel that is independent of the size of the column.

This effectually means that molecules with molecular masses ranging

below the exclusion limit of a gel will elute from a gel in the order of
their molecular masses with the largest eluting first.
Size exclusion chromatography

Smaller Proteins

Bigger Proteins

34
 Gel filtration chromatography
Also called molecular sieve chromatography.
How does it work? If we assume proteins are spherical

size Molecular mass

(daltons)
10,000

30,000

100,000

35
 Gel filtration chromatography

flow

36
 Gel filtration chromatography

flow

37
 Gel filtration chromatography

flow

38
 Gel filtration chromatography

flow

39
 Gel filtration chromatography

flow

40
Gel Filtration Chromatrography
GENERALIZED GFC SETUP

Buffer reservoir
Column
Sample
injector

Pumps Fraction collector

41
 Molecular mass determination by gel filtration
chromatography.
Page 138

Relative elution volume vs. log of molecular mass for variety

of proteins.
 Ion Exchange Chromatography
Separates protein molecules according to their
molecular charge.
In this technique, the beads of the column have a specific charge
on them.( This is a result of a molecule that is attached to these
beads).
The beads might be +ve charged by attaching them to DEAE
(diethylaminoethyl) cellulose or ve charged by attaching
them to carboxymethyl cellulose.
The beads in the column depend on the protein that you want to
purify.
If the protein is ve charged then you have to
use positively charged beads and vice versa.
43
 Ion Exchange Chromatography
How does it work?
For example, if the protein of interest is (- )
negatively charged, then you will use a DEAE-
cellulose column.

The protein will bind to the positively charged beads.

This protein that is attached to the beads can be
released by increasing the concentration of NaCl (or
other salt).

The Na+ ions (or other cation) will compete and bind
Example of cation exchange
to the beads in the column instead of the protein. chromatography
Proteins that are highly( +)positively charged will
emerge first because they will be repelled by the
beads.
44

Protein at pH higher than pI (by at least 1
unit) is negatively charged and binds to
anion-exchange column.

Elution is with increasing [Cl -] (NaCl) or by

decreasing pH (harder to control)

IEC Rules:

- If a protein is most stable below its pI, a

cation exchanger should be used
- If a protein is most stable above its pI, an
anion exchanger should be used
- If stability of the protein is known to be
good over a wider pH range then either
type of ion exchanger can be used
45
 Ion exchange (anion or cation )

Anion-exchange
+
+ +
+ + NaCl
eluent

+
+ Salt gradient
DEAE
+
column
+
+
+
+

+ CH2-CH3
CH2-CH2-N Most negatively charged protein
CH2-CH3
comes off last 46
 Ion-Exchange chromatography
- - + +

If pH mobile phase =7.2

Then charge of the proteins: (-) (-) (+)
(+)
- +
+
++ + + - - -
++ -
+ +
+ -
+ +
+
+ +
+
+
47
Anion exchange column = + charged
 Ion-Exchange chromatography
- - + +

+ + + + Cl-
+
Cl-
+
-
+ + +Cl-
-
-
+ + +Cl-
+
+ Na+
Na+-
+Cl-
Na+ Na+
-
Na+
Na+
- Na+-
Na+
- + Na+ Cl-
+

Increased salt concentration 48

 Anion
Exchanger

Cation
Exchanger

49
 Affinity Chromatography
Uses specific binding properties of molecules/proteins
Stationary phase has a polymer that can be covalently linked to a
compound called a ligand that specifically binds to protein

50
Affinity Chromatography
Other proteins can be separated by this method based on
their affinity for specific groups or compounds.
Examples:

Antigen Antibody

Antibody Antigen

Substrate Enzyme

Concavalin A Glycoprotein

Hormone Binding Protein/Receptor

51
Immuno-Affinity Chromatography
Antibody fixed to
matrix
Protein binds to
antibody
Wash unbound and
loosely bound
proteins off column
Elute protein with
change in salt/pH
52
 Proteins contain hydrophobic amino acid side-chains, some
of which are exposed at the surface of the protein.
Proteins will therefore often bind to other hydrophobic
molecules. Hydrophobic interaction columns are produced
by covalently attaching hydrophobic molecules such as acyl
chains or phenyl groups to insoluble carbohydrate resins.

Similar to ion-exchange chromatography except that column

53
material contains aromatic or aliphatic alkyl groups
Assays, Quantitation and Documentation
Assay enzyme activity at every step
Assay total protein
Run an SDS gel to visualize specific contaminants

54
1-Monitoring Progress of Purification
Protocol
Total protein (mg)
Quantity of protein present in fraction

Total activity (units of activity)

Use a portion of sample to determine activity.

Multiply activity by total volume to determine

total activity.
 Specific activity (units of activity/mg)
S.A. = Total activity
Total protein

% yield: measure of activity retained after each

step in procedure.
% yield = Total activity at particular step
Total activity of initial extract

Purification level: Measure of increase in purity of

protein(enzyme) throughout procedure.
Purification = Specific activity at particular step
Specific activity of initial extract
 Monitoring Progress of Purification
Protocol
Total protein Total activity Specific activity Purification
Step Yield (%)
(mg) (units) (units/mg) level

Initial extract 15,000 150,000 10 100 1

(NH4)2SO4
4,600 138,000 30 92 3
precipitation

Ion-exchange 1,278 115,500 90 77 9

Size
68.6 75,000 1,100 50 110
exclusion
Affinity
1.75 52,500 30,000 35 3,000
column

% yield = Total activity at particular step

Total activity of initial extract
Purification = Specific activity at particular step
Specific activity of initial extract
 Protein detection methods

SDS-PAGE
Visual confirmation

UV Spectrophotometry
Absorbance @ 280 nm
Due mostly to Trp
[Protein] calculated with Beers Law

Colorimetric Techniques
Color change proportional to [protein]
Bradford, Lowry, BCA
58
J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29
Protein Measurement (Colorimetric Techniques) :
1- The Bradford Assay (Coomassie Blue Binding)
Absorbance shift in Coomassie Brilliant
Blue G-250 (CBBG) when bound to
arginine and aromatic residues .
The anionic (bound form) has absorbance
maximum at 595 nm whereas the cationic
form (unbound form) has and absorbance
maximum at 470 nm .

59
More Protein
Protein Measurement:
The Bradford Assay (Coomassie Blue Binding)

Build a Standard Curve

Protein Variation is Great
60
Protein Measurement:
2- The Bicinchoninic Acid (BCA) Assay
The first step is a Biuret reaction which reduces Cu+2
to Cu+1
In the second step BCA forms a complex with Cu+1
which it purple colored and is detectable at 562 nm

61
 3-Lowry assay (Cu reduction)
The first step is a Biuret reaction which reduces Cu+2 to Cu+1
The second reaction uses Cu+1 to reduce the Folin-Ciocalteu
reagent (phosphomolybdate and phosphotungstate).
This is detectable in the range of 500 to 750 nm

4-Absorbance at 280nm
Monitors the absorbance of aromatic amino acids, tyrosine and
tryptophan or if the wavelength is lowered, the absorbance of the
peptide bond.
Purification schemes

63
 Molecular mass
Electrophoresis
Matrix-assisted laser desorption-ionization-time of
flight (MALDI-TOF)
Gel Filtration

Isoelectric point
Isoelectric focusing

3-D Structure
X-ray crystallography

64
64
 Protein Characterization

Characterization of proteins and peptides involves

three different processes:
1. Determining the Amino Acid Composition
- Involves finding out the amino acids that make up
the protein and their number.
2. Determining the Amino Acid Sequence
- Involves finding out the sequence of amino acids
of the proteins in their order.
3. Determining the Molecular mass of the Protein

65
 Primary Structure Determination
How is 1 structure determined?
1) Determine which amino acids are present (amino
acid analysis)
2) Determine the N- and C- termini of the sequence
(a.a sequencing)
3) Determine the sequence of smaller peptide
fragments (most proteins > 100 a.a)
4) Some type of cleavage into smaller units necessary

66
 Amino Acid Analysis
Determines amino acid composition of a protein.
A protein is hydrolyzed in 6N HCl, 24 hrs at 100oC.
Separation of AA by ion exchange chromatography.
 1-Determination of Amino Acid
Composition
They are measured by reacting them with a compound called
ninhydrin. Alpha Amino acids will be given an intense blue color
while imino acids (proline, hydroproline) will be given a yellow
color.
The concentration of a single amino acid is proportional to the light
absorbance of the solution after adding ninhydrin.
The elution profile of the amino acids is obtained. Eluting is the
separation, by washing, of one solid from another.
The identity of the amino acid is revealed by its elution volume,
which is the volume of buffer needed to remove the a.a. from the
column.

68

:
Auto sampler
separation column
reaction
coil reaction
photometer

Coil reaction


130 C

570


Proline & hydroxyproline
440

Photometer
Spectrophotometer
570 440








) (
Amino Acid Analysis

73
 Hydroxyproline

proline
 Sangers Method
O O
N+
F

O
N+
O
Sanger's Reagent

1-Fluoro-2,4-
dinitrobenze
ne

The identity of this 2,4-DNB amino acid derivative is established by chromatography.

75
 Peptide Sequencing: Edman
1.
Degradation
Method for determining N-terminal amino acid.
2. Has been automated.

76
 N C S

Phenylisothiocyanate

- Determining one residue at a time from the N-

terminus
(1) Treat peptide with phenylisothiocyanate (PITC) at pH
9.0 which reacts with the N-terminus to form a
phenylthiocarbonyl (PTC)-peptide.
(2) Treat the PTC-peptide with anh. trifluoroacetic acid
(TFA) to selectively cleave the N-terminal peptide bond
and form a triazolinone derivative.
(3) Extract N-terminal derivative from the peptide.
(4) Rearrange to a phenylthiohydantoin (PTH)-amino acid
with aq. HCl then chromatograph.
77
Peptide Sequencing

78
Step 2: Partial Hydrolysis of Peptides and
Proteins
Carboxypeptidase selectively cleaves the
peptide bond to the C-terminal amino acid.

C-Terminus
H O H O
H
N Protein N
H N OH

R1 H O R2

N-Terminus
this amide is hydrolyzed
by carbopeptidase
79
 Determination of Amino Acid Sequence
- (with enzymes )
Longer polypeptide chains are broken into shorter ones for analysis
by specifically cleaving them with enzymes that cleave at specific
points.
Some examples are :

Enzyme Cleavage Point

Trypsin Lys, Arg (C)
Chymotrypsin Phe,Trp, Typ (C)
Pepsin Phe, Trp, Tyr (N)
Cyanogen bromide Met (C)

80
Primary Structure Determination

81
Peptide Digestion

82
 Determining Protein Sequence
After cleavage, mixture of peptide fragments produced.
Can be separated by HPLC or other chromatographic techniques
Use different cleavage reagents to help in 1 determination

83
Putting it all together!
Suppose an unknown hexapeptide gave tagged A
(alanine) upon treatment with Sangers reagent, and
upon treatment with carboxypeptidase, the first amino
acid released was M (methionine) followed by G
(glycine)
Partial hydrolysis gave the following identifiable
tripeptides: V-G-M, A-S-F, and S-F-V. What is the
1 structure of the hexapeptide? A S F V G M

A S F V G M
V G M
A S F V G M
84
85
86
Mass spectrometry:

Molecules are vaporized and ionized, and the

degree of deflection (mass-dependent) of the ions in
an electromagnetic field is measured
extremely accurate, but expensive
MALDI can measure the mass of proteins smaller than
100 KDa

Helpful to detect post-translational modification

Protein sequencing: relying on the protein data base

87
Protein Characterization
3. Determination of Molecular Mass by
Mass Spectrometry Diagram

88
Time-of-flight (TOF) mass
spectrometry
 Staining
Stainingtotofind
find
-how the cells
-how the cells Staining
Stainingtotofind
find
look
look(anatomy)
(anatomy) -if
(TARA) -if cells are deadoror
cells are dead
(TARA) alive
alive(viability)
(viability)
TISSUE (TUMUL)
CELLS (TUMUL)

Histology

Cell Biology

Proteins

Biochemistry
lo gy
Bio
Western
WesternBlotting c ular DNA
Blotting le
-To
-To detectproteins
detect proteins Mo
ELISA Southern Blotting
ELISA Southern Blotting
-To
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quantifyproteins
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findcopy
copynumber
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NorthernBlotting
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Enzyme
EnzymeAssays Real-Time PCR Genome
Assays Real-Time PCR GenomeSequencing
Sequencing
-To
-To measureenzyme
measure enzymeactivity
activity RNA -To
-Tostudy
studygene
geneexpression
expression
90
(TUMUL, BASIA
& MYSELF)
 ProteinCharacterization
Immunological methods:
Can be used to detect or to quantify proteins
Antibodies
Use protein of interest to raise antibodies (rabbit)
Different antibodies can recognize different regions (epitopes)
Can distinguish differences as small as 1 residue
Attachment of indicators- dyes, radioactivity
Applications- e.g.

- immunoassay,
- Western blotting (antigen detection)
- ELISA = Enzyme-linked immunosorbent assay (antigen
quantification)
91
91
 Using antibodies

Antibodies (immunoglobulins) bind specific antigens/epitopes

monoclonal - all bind same epitope
polyclonal - mixture that binds several epitopes
Secondary antibodies - anit-immunoglobulins (antibodies to antibodies)
92
 Electrophoresis
Western blot analysis
This is a powerful application of SDS-
PAGE;

Allows the detection of a single protein of

interest present in a complex mixture of
proteins;

Antibodies:
4 chains: 2 light chains (25 kDa ea)
and 2 heavy chains (50 kDa each);

Structure of an antibody

93
 Electrophoresis
Western blot analysis
Western blot analysis can detect one protein in a mixture of any
number of proteins while giving you information about the size of
the protein

94
 Western Blotting (WB)
WB is a protein detection technique that combines the
separation power of SDS PAGE together with high
recognition specificity of antibodies .
An antibody against the target protein could be purified from
serum of animals (mice, rabbits, goats) immunized with this
protein.
Alternatively, if protein contains a commonly used tag or
epitope, an antibody against the tag/epitope could be
purchase from a commercial source.

95
 WB, Steps 3-4: Detection
fluorochrome

)Fluorescein isothiocyanate(FITC

96
 Quantifying antigen by
Enzyme-linked immunosorbent assay
(ELISA)

ELISA uses enzyme to detect an antigen, by building

up layers of reactants.

Several variants:

capture assay.

sandwich assay.

ELISA gives quantitative information - tells us the
concentration of protein (relative to a standard).

97
97
The ELISA (Enzyme Linked Immunosorbent Assay)
is an immunotest with high sensitivity, in which the antigen or
the antibody is linked to a solid (plastic) surface.
The test generally performed in plastic plates with 96 wells (in
100-200 l volume).
Mostly, the the antigen is pre-absorbed to the plastic surface
then different dilutions of the tested serum sample (from a
patient) are added to the wells.

Application: The application scale of ELISA is broad (e.g.

Identification of viral and bacterial infections; the
identification and quantification of hormones or cytokines in
blood circulation, etc).
The antibodies produced against pathogenic microorganism
can be identified in blood. An infection state can be deducted
from the increase or decrease of specific antibodies.
The test sensitivity is high, ng amount can be measured.
98
 Capture
Substrate Colour

enzyme

antibody -enzyme

antibody
antibody

antigen
99
99
 ELISA data from three patients

Positive Negative Patient Patient Assay

Control Control Patient A B C Control

1.689 0.153 O.055 0.412 1.999 0.123

100
 YY Y YY Y
Wellwithantibodies
WellwithantibodiesandBSAadded YY Y
Wellwithantibodies,
antigen binding sites BSA,andtestsample

1Wellinamicrotiterplate

Antibodystructure
YY Y
Wellafterwasheswith
washbuffer

Wellafteraddingsubstrate

Colordevelopedduetothe
formationofasubstrate YY Y
absorbance is read by Secondaryantibodylinkedto
fiberoptic multichannel anenzynmeisaddedtothewell
spectrometer. YY Y
Wellafterremoving
101
101
excessantibody
 Example :
At pH 7.5 of the mobile phase to be used on the columne,
peptide A has a net charge of 3 (presence of more Glu a
Asp residues). Peptide B has net charge +1. Which peptide
would elute first from cation-exchange resin? Which peptide
would elute first from anion-exchange resin?
A cation-exchange resin has negative charges and binds
positively charged molecules B will be retarded and
A will elute first
An anion-exchange resin has positive charge and binds
negatively charged molecules A will be retarded

B will elute first

102
 Determination of protein sequence
1. Enzyme mapping example 2
The following data were obtained after treating an octopeptide with the
following reagents:

HCl 6M: Ala, Gly2, Lys, Met, Ser, Thr, Chymotrypsin: 2 peptides were
Tyr obtained:
Peptide 5: Gly, Tyr
Peptide 6: Ala, Gly, Lys, Met, Ser, Thr
CNBr: 2 peptides were obtained:
Peptide 1: Ala, Gly, Lys, Thr
Peptide 2: Gly, Met, Ser, Tyr FDNB: yields Gly

Trypsin: 2 peptides were obtained: Carboxypeptidase A: yields Gly

Peptide 3: Ala, Gly
Peptide 4: Gly, Lys, Met, Ser, Thr, Tyr

What is the sequence of this peptide?

103
Thank you..

104