Analytical techniques for separation or purification of proteins

Analytical techniques for
separation or purification of proteins
Dr. Rohini C Sane

Applications of Separation of proteins (purified proteins)
❖Applications of Separation of proteins ( purified proteins):
1. Quantitative analysis
2. Therapeutic / clinical use
3. Research purpose

Properties of proteins used for their Separation
❖Separation of proteins from other proteins and molecules is achieved by
combination of several methods , based on Properties such as :
1. Solubility
2. Molecular size
3. Molecular charge
4. Specific binding of the protein to the specific substance

Separation of proteins based on Solubility
❖Separation of proteins based on Solubility : the Solubility of protein is
dependent of the salt concentration of the solution.
❖The Solubility may be :
a. Increased at a low salt concentration called salting in or
b. decreased at high salt concentration called salting out
➢ when a salt like ammonium sulphate is added to the solution , some
protein precipitate at a given salt concentration , while other remain in
solution.
e.g. serum immunoglobulins are precipitated by 33 – 40% saturated
ammonium sulphate solution while serum albumin remains soluble at this
concentration and thus we can separate it.

Schematic diagram for Full saturation and half saturation for fractionation of proteins
Full saturation : Half saturation:
High(Globulins) & low (Albumin )
molecularweightproteinsgetprecipitated
Low molecular weight
proteins in the
supernatant
e.g. Albumin with mw
60000
High molecular
Weight proteins
get precipitated
e.g. Globulins
with mw
180000
Application:Fullsaturationandhalfsaturationareuseful forfractionationofproteins
frommixture(e.g.separatingalbuminandglobulinfromserumproteins).

Separation of proteins based on molecular size
❖On the basis of molecular size, proteins can be separated by:
1. Dialysis
2. Gel filtration ( molecular sieving)
3. Ultracentrifugation

Dialysis for Separation of proteins based on molecular size
❖Principle of Dialysis : Small molecules ,such as salts can be removed
from protein solution by dialysis using semipermeable membrane
tube/bag.
❖Process of Dialysis : it is performed by adding/ passing protein salt
solution to a semipermeable membrane tube/bag (cellophane bag).
When tube is immersed in a dilute buffer solution ,small molecules will
pass through and large molecules can be retained in the tube, depending
on the pore size of the dialysis membrane .
❖Biochemical Application of Dialysis : for removal of ammonium sulphate
solution ( desalting)during protein purification
❖Clinical Application of Dialysis : management of kidney failure patients

Process of Dialysis for Separation of proteins based on molecular size
Itisperformedbyadding/passingproteinsaltsolutionthroughasemipermeable
membranetube/bag(cellophanebag).Whentubeisimmersedinadilutebuffersolution
,smallmoleculeswillpassthroughandlargemoleculescanberetained inthetube,
depending ontheporesizeofthedialysismembrane.

Principle of Gel filtration (molecular sieving) for Separation of proteins
based on molecular size
Principle of Gel filtration : the rate at which a molecule flows through the
column is dependent on its size and shape. Small molecules enter the pores of
the gel molecule but larger molecules cannot. Therefore, large molecules take a
more direct route through the column . The volume of solvent available for
these is greater than that for the lager molecules. So the small molecules flows
through the column more slowly.

PrincipleofGel filtration for separation of proteins
PrincipleofGelfiltration:therateatwhichamoleculeflowsthroughthecolumnis
dependentonitssizeandshape.Smallmoleculesentertheporesofthegelmoleculebut
largermoleculescannot.Therefore,largemoleculestakeamoredirectroutethroughthe
column.Thevolumeofsolventavailablefortheseisgreaterthanthatforthelager
molecules.

Gels used in Gel filtration(molecular sieving)for Separation of proteins
based on molecular size
❖Gel filtration or gel permeation chromatography uses a column of insoluble ,
but highly hydrated polymers in the form of porous beads. e.g.
1. Dextran (Sephadex series)
2. Agarose (Sepharose series)
3. Polyacrylamide (Bio Gel P series)

Commercially available gels
❖Commercially available gels :
• Sephadex : G-10,G-100
• Biogel : P-10, P-30,P-100
• Sepharose : 2B, 4B,6B

Technique of Gel filtration (molecular sieving) for Separation of
proteins based on molecular size
• Gel filtration / molecular sieve chromatography/ molecular chromatography:
is a technique used to separate solutes based on their molecular size .
• The sample solution containing a mixture of proteins is passed through a
column of polymeric material previously equilibrated with the buffer.
• Gels used to prepare column : cross- linked dextran (e.g. Sephadex), Agarose
(e.g. Sepharose), polyacrylamide , polystyrene
• Proteins of different molecular size penetrate into the internal pores of the
gel to varying degrees .
• Large Protein molecules that cannot enter the pores remain in the excluded
volume , whereas small Protein molecules enter the pores. Protein of
intermediate size are excluded based on their size.

Small molecules
Large molecules
Smaller
molecules
enter the
gel matrix
but larger
molecules
cannot
enter the
pores of
the gel
Gel polymer
Sample containing a mixture
of large and small protein
molecules loaded into the
column.
Larger
molecules
are eluded
out of the
column .
molecular
sieving
Schematic diagram of Gel filtration (molecular
sieving) for separation of proteins based on
molecular size
v

Applications of Gel filtration or gel permeation chromatography
❖Applications of Gel filtration or gel permeation chromatography :
1. Determination of molecular weight of proteins ( using a calibrated column
with a calibrated column with molecular weight markers)
2. Separation of proteins
3. Protein purification
4. For desalting protein solution

Principle of Ultracentrifugation and its applications for
isolation of proteins/ enzymes
❖Principle of Ultracentrifugation: is based on the generation of centrifugal
force to as high as 600000 g (earth’s gravity g= 9.81 m/s2)that allows the
sedimentation of particles or macro molecules.
❖Applications of Ultracentrifugation : is a tool for isolation of subcellular:
1. Proteins
2. Enzymes
3. Organelles
4. Nucleic acids
➢ Ultracentrifugation is employed in determination of molecular weight of
macromolecule.

UltracentrifugationforSeparationofproteinsbasedonmolecularsize
❖Ultracentrifugation for Separation of proteins based on molecular
size : High speed centrifugation can separate a protein solution in
various components . The rate at which a protein can sediment in a
centrifugal field depends on its :
1. Size
2. Shape
3. Molecular weight
For proteins of similar shape, centrifugal force sediments those
molecules having large molecular weight .
Centrifugalforceisexpressedintermsofsedimentationcoefficient(S)=v/
2r

Sedimentationcoefficient(S)ofproteins
❖Sedimentation coefficient (S) = v/ 2r
v = migration (sedimentation) of themolecule
= rotation of centrifuge rotor in radians/ sec
r= distance in cm from the Centre of rotor ( radius of rotor)
Units of Sedimentation coefficient in seconds : Svedberg Unit
❖Sedimentation coefficient of :
1. Hemoglobin = 4x 10 – 13 s or 4S
2. Ribonuclease = 2 x 10 – 13 s or 2S
3. Prokaryotic Ribosome 70 S = 70 x 10 – 13 s
4. Eukaryotic Ribosome 80 S = 80 x 10 – 13 s

Schematic representation of Ultracentrifugation used for
Separation of proteins based on molecular size
❖The rate at which a protein
can sediment in a
centrifugal field depends on
its Size , Shape and
Molecular weight .
❖For proteins of similar
shape, centrifugal force
sediments those molecules
having large molecular
weight .

Technique for separation of cellular proteins by ultracentrifugation
PreparationofHomogenate-Celldisruptionbysonication/osmoticshock/homogenizerin(0.25M)Sucrose
Ultracentrifugation at 700 g X 10 minutes
Separation of supernatant I and nuclear fraction
Separation of supernatant II and mitochondrial fraction
Separation of supernatant III and microsomal fraction

Separation of subcellular organelles /protein factions by differential ultracentrifugation
Homogenate
supernatant I
nuclear fraction
supernatant II
mitochondrial fraction
supernatant III
microsomal fraction
❖ PreparationofHomogenate:The cells are subjected to
disruption by sonication/ osmotic shock /
homogenizer in (0.25M)Sucrose.
❖ Advantage of sucrose medium : it dose not cause
organelles to swell . The subcellular organelles can
be separated by differential ultracentrifugation .
❖ Major cell fractions by commonly employed
method : 1.nuclear fraction
2.mitochondrial fraction
3. microsomal fraction
➢ Microsomes : are small vesicles formed from
rough endoplasmic reticulum( studded with
ribosomes) during the process of cell
fractionation .
The Microsomes as such do not occur in the cell.
Schematic diagram

Maker enzymes to check purity of subcellular fractions
To check purity of subcellular fraction Maker enzymes
Nuclear fraction DNA polymerase
Mitochondrial fraction Glutamate dehydrogenase
Ribosomal Glucose 6-phosphatase
Cytosolic fraction Hexokinase

Chromatography for separation of Proteins
❖Chromatography( chroma= color, graphein =to write): is one of analytical
technique dealing with separation closely related compounds from a
mixture of proteins ,peptides and amino acids ( also lipids, carbohydrates,
vitamins ,drugs).

Classification Chromatography
Chromatography
Partition
Paper
chromatography
Single
dimensional
Ascending
Descending
Two dimensional
Thin layer
chromatography
Gas
chromatography
Adsorption
Column
TLC
Ion- exchange Gel filtration Affinity HPLC
❖ Basis of classification of chromatography:
a. the physicochemical interactions(Adsorption/
partition/ Ion-exchange/ Molecular-sieving/
Affinity)
b. the nature of the stationary phase ( paper ,thin
layer, column)
c. the nature of both the stationary phase and the
mobile phase( gas- liquid chromatography)

Applications of Chromatography technique
1. Used for separation of Proteins/peptides ,amino acids, carbohydrates
2. Purification of proteins ,enzymes , immunoglobulins , membrane
receptors , nucleic acids
3. Determination of molecular weight of a protein
4. Analysis of hormones , brain amines ,vitamins, drugs
5. Qualitative and quantitative analysis of complex mixtures
6. In Clinical laboratory for the identification amino acids (amino acidurias),
peptides/proteins, sugars ,drugs in serum or urine or biological fluids

Principles of Chromatography used for separation of Proteins
❖Principles of Chromatography : it consist of mobile phase and stationary phase.
❖Mobile phase : refers to the mixture of substances (to be separated) dissolved in
liquid or gas
❖Stationary phase : a porous solid matrix through which the sample contained in
the mobile phase percolates
➢Interactions between stationary phase and mobile phase results in the separation
of the compounds from the mixture of proteins ,peptides and amino acids.
➢These interactions include the physicochemical principle such as:
1. Adsorption
2. Partition
3. Ion-exchange
4. Molecular –sieving
5. Affinity

Principle of Paper Chromatography technique for separation of proteins/
peptides
• Paper Chromatography : is a partition type of chromatography .
• Principle of Paper Chromatography technique for separation of proteins/
peptides : separation of mixture of proteins/ peptides is effected by the
continuous partition of the proteins/ peptides between stationary water
phase adsorbed on the Whatmann filter paper number 3 and moving organic
solvent flowing over the filter paper(mobile phase) .
• The separation depends on the relative tendencies of the molecule in a
mixture to associate more strongly with one or the other phase.
• Solvent system provides both stationary phase and mobile phase .
• Mobile phase : an organic solvent layer of mixture of Butanol : Acetic acid :
Water in proportion of 4:1:5 v/v

Technique of Paper Chromatography for separation of proteins/ peptides
❖Technique of Paper Chromatography for separation of proteins/ peptides :
1. A small amount of the mixture of proteins/ peptides (in solution) to be separated is applied on
the filter paper( Whatmann 1 or3 ) at one point on a pencil line near lower edge along with
known standards with the help of fine capillary .
2. Samples are spotted on the same line at the distance of 2 cm apart from each other with the
known standards .
3. The paper is supported vertically so that the edge near the pencil line(closed to spots) is dipping
in organic solvent ( taken in a trough).
4. A bell-jar is kept covered the paper and the trough.
5. The organic solvent (mixture of Butanol : Acetic acid : Water in proportion of 4:1:5v/v) rises
slowly in upward direction and it carries each amino acid of mixture and standard amino acids.
6. Sufficient time is allowed for the solvent to reach almost the opposite edge of the paper. The
bell- jar is removed. The paper is then removed.
7. Quickly mark the edge of solvent with pencil .This is known as a solvent front .
8. The solvent is allowed to evaporate from the filter paper . The filter paper is then sprayed with
appropriate staining solution.

Ascending and descending paper Chromatography for separation of Proteins
Ascending chromatography:
migration of solvent upwards
Descending chromatography:
migration of solvent downwards
•••
Mobile phase
Mobile phase
Stationary phase ( paper strip)
• • •

Two dimensional chromatography for separation of proteins
❖Sometimes ,it is rather difficult to separate a complex mixture of
biomolecules by a single run with one solvent system .
❖Two dimensional chromatography: enhances the separation of a mixture
into individual components .
• In such a case, second run is carried out by different solvent system , in a
direction perpendicular to the first run after drying the paper. This is
referred as Two dimensional chromatography.
• After location a map obtained and
the compounds can be identified by
comparing their positions with a map
of known compounds under same
conditions .
Direction of first run of chromatographyDirectionof
secondrunof
chromatography

Circular paper Chromatography for separation of Proteins
• If a Circular paper is used with solvent entering the paper from its centre and
the sample applied close to the centre on the periphery of the circle , it is
called circular chromatography.

Principle of Thin layer Chromatography technique used for separation of
proteins/ peptides
• Principle of Thin layer Chromatography for separation of proteins/ peptides :
separation of mixture of proteins/ peptides is effected by the continuous
partition of the proteins/ peptides between stationary water phase adsorbed
on the inert substance such as cellulose spread as a thin layer on glass or
plastic plates and moving organic solvent flowing over the filter paper( mobile
phase) .
• Mobile phase : an organic solvent layer of mixture of Butanol : Acetic acid :
Water in proportion of 4:1:5v/v
• Adsorption thin layer chromatography : adsorbents such as activated silica
gel, alumina , kieselguhr are used.
• Gas chromatography (GC) and gas liquid chromatography(GLC) for separation
of proteins/ peptides : where the mobile phase is either gas or liquid gas, the
basic principle remaining the same.

ThinlayerChromatographytechniqueforseparationofproteins/peptides
Principle of Thin layer Chromatography for separation of proteins/ peptides :
separation of mixture of proteins/ peptides is effected by the continuous partition of the proteins/ peptides
between stationary water phase adsorbed on the inert substance such as cellulose spread as a thin layer on
glass or plastic plates and moving organic solvent flowing over the filter paper( mobile phase) .

Advantages of Thin layer Chromatography technique for separation of proteins/
peptides over paper Chromatography
• Advantages of Thin layer Chromatography technique for separation of
proteins/ peptides over paper Chromatography :
1. Rapid process :separations can be completed within an hour.
2. Require small sample volume
➢Other applications of Thin layer Chromatography technique:
a. Identification of drugs in case of poisoning
b. Determination of lecithin /sphingomyelin ratio in respiratory distress
syndrome
c. Separation of neutral lipids and phospholipids

Principle of Ion-exchange chromatography for Separation of peptides/proteins
❖Principle of Ion-exchange chromatography : it is a technique by which
molecules are separated based on their electrical charges (differences
in acid-base behavior) e.g. separation of proteins.
• The separation are carried out in columns packed with an ion-
exchangers.
• Anionexchangeresinsexchangesitsanion(A-)withanotheranion(B-)in solution:
R+A- + B-  R +B- + A-
• Cation exchange resins exchanges its cation (H+) withanothercation(C+)in
solution :
H+R- + C+  C+R- + H+

Comparison of Anion and Cation exchanger resins used in ion-exchange chromatography
Anion exchanger resins Cation exchanger resins
Contain fixed positively charged groups
and attract negatively charged anions
(to be exchanged)
Contain fixed negatively charged
groups and attract positively charged
cations(to be exchanged)
Also called basic ion exchangers Also called acidic ion exchangers
Strong anion exchangers are resin
containing substituted with tertiary
amines e.g. diethyl aminoethyl (DEAE)
Sephadex
Strong cation exchangers are resin
containing substituted with sulfonic
acid groups e.g. ( a sulphonated
polystyrene -Dowex -50)
Weak anion exchangers are resin
containing substituted with secondary
amines e.g. (DEAE- cellulose )
Weak cation exchangers are resin
containing substituted with
carboxylate groups -CarboxyMethyl
dextran(CM-Sephadex)

Anion exchanger resins and Cation exchanger resins used in Ion exchange chromatography
for Separation of peptides/proteins
Anion exchanger resins : contain fixed
positively charged groups and attract
negatively charged anions (to be exchanged)
Cation exchanger resins :contain fixed
negatively charged groups and attract
positively charged cations(to be exchanged)

Anionic site
SO3
- Na+
SO3
- Na+ + N
+
H
3
- CHR-COOH  Amino acid
SO3
-N
+
H
3
-CHR-COOH
SO3
-N
+
H
3
-CHR-COOH + 2 Na
+
Resin
particle
cation exchange
Resin
particle
Mechanism of cation exchange in ion exchange chromatography

Technique of Ion-exchange chromatography applied for
Separation of proteins/peptides based on molecular charge :1
❖Ion-exchange chromatography: separation is carried out in the column
(consist of a long tube) filled with Ion exchange resin carrying either anionic
or cationic groups.
❖Anion exchange resins : have fixed positive charge groups and
exchangeable anions e.g. diethylaminoethyl (DEAE) Sephadex
❖Cation exchange resins : have fixed negative charge groups and
exchangeable cations e.g. (Dowex -50), carboxymethyl dextran(CM-
Sephadex), CM(carboxymethyl) cellulose . Strong Cation exchangers are
used for separation of amino acids (amino acid analyzers).
❖The affinity of each peptide /protein molecule for the resin is affected by
pH, which determines the ionization state of the protein molecule.

Technique of Ion-exchange chromatography applied for
Separation of proteins based on molecular charge :2
❖Procedure : Mixture of protein which is to be separated is applied /poured on
the column packed with resin and allowed to percolate through slowly . At the
appropriate pH , the charged protein groups bind to oppositely charged
groups of resin by ionic interactions . Thus, proteins are bound to the ion
exchange resin in the column. The tightness of the binding of a particular
protein depends on how many charged moiety are available to interact with
the ion exchanged resin.
❖Technique of elusion of protein from the ion exchanger : by washing /eluting
with a salt solution that disrupts the electrostatic interactions of the protein
and ion exchange resin. Gradual increase in salt concentration is applied to
column , when weakly bound proteins are eluded first followed by tightly
bound proteins .

Technique of Ion-exchange chromatography applied for Separation of proteins
based on molecular charge :3
Ion-exchangechromatography:thecolumnconsistofalongtubefilledwithIonexchange
resincarryingeitheranionicorcationicgroups.Theaffinityofeachproteinmoleculeforthe
resinisaffectedbypH,whichdeterminestheionizationstateoftheproteinmolecule.

Schematic diagram for steps of separation of proteins/ amino acids in Ion-exchange
chromatography
Ion exchange chromatography: separation is carried out in the column (consist of a long
tube) filled with Ion exchange resin carrying either anionic or cationic groups.

Applications of Ion-exchange chromatography
1. Purification of proteins , enzymes, hormones, antibodies (also nucleic acid and
polysaccharides)
2. Separation of amino acids using strong Cation exchangers (amino acid analyzers)
3. Determination of molecular weight of proteins /macromolecules
4. Desalting of low molecular weight compounds during protein purification
5. Concentration of solution containing high molecular weight compounds. Water and low
molecular weight compounds are absorbed by the gel , high molecular weight
compounds that remain in solution become more concentrated without any change in
their pH or ionic strength.
Other applications :
a. Preparation of deionized water
b. Preparation of ultrapure, metal –free reagents
c. Separation of vitamins

Principle and Technique of Affinity chromatography
❖Principle of Affinity chromatography : is a technique for separating proteins /
macromolecules based on the specific affinity (non covalent binding) of a molecule
for its ligand (such as an enzyme for its substrate/ cofactor or protein for its
antibody) which is attached to gel matrix .
❖Technique of Affinity chromatography :
➢The sample (mixture)to be separated is applied to a column of ligand bound to a
matrix such as crossed-linked dextran or agarose (Sepharose).
➢ The molecule that has high affinity for the ligand binds to it ,while other molecules
are eluted out of column. The immobilized ligand acts as molecular fishhooks to
selectively pick up the desired protein while the remaining proteins pass through
the column.
➢ The bound molecule is eluted by changing the conditions (change in pH or salt
concentration) so that binding /affinity is does not exist(break protein-ligand
interaction) .

Schematic diagram for Technique of Immunoaffinity chromatography
immobilized antibody covalently
attached to matrix
Application of a
sample containing
target Antigen
and non-specific
proteins
elution of non-
specific proteins
changing pH or
salt concentration
elution of a specific protein
by changing pH or salt
concentration
Binding of antigen to immobilized
antibody after application mixture of
proteins on a column
Regeneration of column
Inert and porous matrix

Diagrammatic representation for Technique of Affinity chromatography

Types of Affinity chromatography
❖Types of Affinity chromatography :
Criteria Immunoaffinity
chromatography
Pseudo affinity
chromatography
Metal
chromatography
Immobilization of Biological molecules
and small molecules
(ligand bound to it) .
e.g. An antibody is
used as the
immobilized
molecule to capture
its antigen ( vice
versa) .
Compound that
resembles a
biological ligand on a
solid material and
enzymes or proteins
bound to it .
A metal ion on a
solid support and
binds to specific
metalloproteins

Applications of Affinity chromatography
1. Purification of proteins , enzymes , immunoglobulins , antibodies,
membrane receptors (also nucleic acid and polysaccharides)
2. Isolation of glycoproteins by lectins
3. Purification of immunoglobulins from serum or cell extracts by protein A –
agarose
4. Purification of vitamins ,nucleic acids ,drugs

Applications of Affinity chromatography
Applications of
Affinity
chromatography
Purification of proteins ,
enzymes , immunoglobulins ,
antibodies, membrane receptors
(also nucleic acids, vitamins and
polysaccharides)
Purification of
immunoglobulins from
serum or cell extracts by
protein A –agarose
Isolation of
glycoproteins by
using lectins

Principle and Instrumentation High performance liquid chromatography(HPLC)
❖Principle : is a chromatographic technique carried out at high pressure using columns packed with
tiny beads of matrix(non-compressible resin material) and automated equipment that enables
highly reproducible profiles.
❖Instrumentation : the components in HPLC include :
1. Solvent reservoir made of stainless steel to store mobile phase.
2. A high pressure pump to drive sample and mobile phase( solvent ) through tightly packed
column of adsorbent to give constant flow . The pump may be a holding coil ,pneumatic
amplifier , piston or syringe.
3. Sample injection device that may be a micro syringe or a small volume metal loop.
4. A column made up stainless steel in which separation takes place .Eluent does not flow through
the column under the force of gravity but hydrostatic pressure 5000 to10000psi (pounds
/square inch) used in routine separation .
5. Different types Detector : eluants are detected by UV adsorption, fluorescence ,or
electrochemical, variable wavelength , scanning wavelength,
6. Recorder
❖Applications of HPLC : for quick and satisfactory fractionation of proteins and peptides

Components of Instrument used in High performance liquid chromatography
Schematic diagram
Solvent reservoir high pressure pump
Sample
injection
loop
Stainless
steel
column
for
separation
of proteins
/ peptides
Detector Recorder
Sample
collector

Applications of High performance liquid chromatography(HPLC)
❖Applications of High performance liquid chromatography(HPLC)
a. for quick and satisfactory fractionation of proteins and peptides
b. Separation of amino acids ,biogenic amines, polyamines ,neuropeptides,
hormones
c. Separation of carbohydrates ,drugs , steroids

Applications of High performance liquid chromatography(HPLC)
Applications of High
performance liquid
chromatography(HPLC)
For quick and satisfactory
fractionation of proteins
and peptides
Separation of
carbohydrates ,drugs ,
steroids
Separation of amino
acids ,biogenic amines,
polyamines,
neuropeptides,
hormones ,

Principle of Electrophoresis for Separation of proteins
❖Electrophoresis : The movement of a charged particles in an electric field towards
the oppositely charged electrodes (anode or cathode) depending on the net charge
carried by the particle . Application of electric field to solution of ions makes ions to
move.
❖Principle of Electrophoresis for Separation of proteins :
❖At pH < PI , the proteins bear net positive charge (a cation) moving towards the
negatively charged pole i.e. cathode
❖At pH > PI , the proteins bear net negative charge (an anion) moving towards the
positively charged pole i.e. anode
❖Rate of migration of protein molecules in electric field  the charge
❖Rate of migration of protein molecules in electric field  1/ size of protein molecules
❖The rate of movement is determined by the charge /mass ratio on protein
molecules placed in a electric field .

Classification of electrophoresis based on the mode of operation and separation
Electrophoresis
Boundary Zone
paper
Cellulose acetate
Gel
Immuno-electrophoresis

Moving boundary electrophoresis
❖Moving boundary electrophoresis :
• described by Tiselius.
• is rarely used.
• The macromolecules are present freely in solution and the separation
occurs only at the boundaries while the bulk of the solution remains
homogenous .

Technique for Moving boundary electrophoresis

Types of supporting media of zone electrophoresis for
separation of serum proteins
❖Zone electrophoresis : the sample is applied as a spot or band on chemically inert and
homogenous supporting medium on which separation of the components takes place in
the form of zones or bands.
❖Types of supporting media/gel used in zone electrophoresis for separation of serum
proteins :
1. Paper : the earliest material to be used , mixing occurs between zones due to
adsorption of the molecules on the cellulose from paper material
2. Cellulose acetate : widely used supporting media in clinical laboratories, serum
proteins separation is completed within an hour, faster than compared paper
electrophoresis, minimal adsorption of molecules and clear separation of mixture
into discrete zones →5 bands
3. Agarose gel: a transparent medium, material of choice for immunoelectrophoresis,
prepared on microscopic slides, suitable for photometric scanning (10-15 bands)
4. Polyacrylamide gel: a transparent medium, fine resolution of complex mixture,
scanning feasible in visible and UV region (20 bands)
5. Starch grains : prepared by compressing whole Starch grains in buffer into blocks .
Large molecules such as isoenzymes are separated using Starch grains.

Comparison of Types of supporting media of zone
electrophoresis for separation of serum proteins
Cellulose acetate
widely used supporting
media in clinical
laboratories
serum proteins separation
is completed within an
hour, faster than paper
electrophoresis
Cellulose acetate shows
minimal adsorption of
molecules and clear
separation of mixture into
discrete zones
serum proteins separated
into 5 bands
Agarose gel
material of choice for
immunoelectrophoresis
a transparent medium
prepared on microscopic
slides
into 10-15 bands
Polyacrylamide gel
fine resolution of complex
mixture
a transparent medium
scanning feasible in visible
and UV region
into 20 bands

Factors affecting migration and the resolution (separation) during
electrophoresis
Medium used in electrophoresis The separation is during electrophoresis
determined by
With large pores ( cellulose acetate , agar,
paper)
Electrical Charge on the molecule to be
separated .
The Fine gel ( acrylamide ,agarose) Size of the molecule to be separated. Gel
acts as a molecular sieve.

Electrophoresis apparatus
❖Electrophoresis apparatus :is designed so that the circuit between the two poles is
bridged by the support medium holding sample and the current flow is partially
carried by the components of the sample .
❖Electrophoresis chamber : consist of two compartments separated from each
other by dividing wall, one side contains anode and other side the cathode (
platinum wire).
❖Each side is filled to the same level with a buffer( barbital buffer pH 8.6) is often
used for serum protein electrophoresis.
❖A “bridge” (Electrophoresis support) across the top of the dividing wall holds a
membrane or other support material so that either end of support material is in
contact with the buffer in corresponding the compartments.
❖The only connection between the two compartments is by through the paper /
membrane.
❖First ,paper /membrane is immersed in buffer , blotted and then sample is applied.
❖When voltage is applied to the cell ,the current is carried across the porous
membrane by the buffer ions.

Schematic diagram of horizontal strip electrophoresis tank : Technique for Separation of
amino acids /proteins
+ _
Electrophoresis support
Filter paper wicks
anode compartment
cathode compartment
Barbiton
Buffer
PH 8.6
Strip-Cellulose acetate /Whatmann No.1paper
Power supply—0.5-10 mA
Stains specific for proteins : Amido black 10B, Bromophenol blue, Azocarmine B

Selection of buffer for electrophoresis
❖Buffer for electrophoresis:
a. Provides desired pH .
b. Must not react with the sample.
c. If the buffer concentration is increased ,the migration rate is decreased ,
but resolution of components is improved .
d. The reduction in sample migration distance is due to competition from
the increased number of buffer ions available to carry the current.
e. As ionic strength of the buffer (the number of available ions)is increased,
more current can be carried. The increased current results in elevated
temperatures which may denature some proteins and affect the
separation .
➢Therefore, selection of proper buffer for electrophoresis is important.

Power supply used in electrophoresis
• A Power pack is used to supply either constant current or constant voltage
during electrophoresis.
• The Power supply used in electrophoresis can be adjusted to provide either
constant current or constant voltage .
• If the voltage is kept constant during the separation of proteins by
electrophoresis, the current will gradually increase, accompanied by an
increase in temperature.
• For short –term electrophoresis constant voltage is often used ,for longer
periods of time, constant current is preferable because temperature does
not increase appreciably during electrophoretic run.

Technique for Separation of proteins using electrophoresis:1
1. The cellulose acetate strip is moistened with a buffer at a fixed/given pH and placed
between cooling plates . Cellulose acetate serves as a matrix to prevent the protein
molecules from diffusing randomly . Cellulose acetate shows minimal adsorption of
molecules and clear separation of mixture into discrete zones .
2. A drop of solution of the protein mixture is applied in the center of cellulose acetate
strip.
3. The ends of the strip are immersed in a buffer from the electrode compartments.
4. Application of direct current electric field separates proteins based on their net
electric charge at the pH used. Rate of migration of protein in electric field depend on
the pH of medium /buffer and acid –base properties of each protein.
5. Proteins that are cations at the pH used will migrate towards the cathode (negative
electrode), anions will move to anode (positive pole) as indicated at time T1.
6. At the end of process, the positions of proteins are made visible with stains that bind to
proteins .

Technique for Separation of proteins using electrophoresis :2
The cellulose acetate strip is moistened with a buffer at a fixed/given pH and placed
between cooling plates .
A drop of solution of the protein mixture is applied in the center of cellulose acetate strip.
The ends of the strip are immersed in a buffer from the electrode compartments.
Application of direct current electric field separates proteins based on their net electric
charge at the pH used.
Proteins thatarecationsatthepHusedwillmigratetowardsthecathode(negativeelectrode), anionic
poteinswillmovetowardsanode(positivepole)asindicatedattimeT1.
At the end of process, the positions of proteins are made visible with stains( Amido Black108,
Bromophenol blue , Azocarmine B) that bind to proteins .

Schematic diagram for Separation of proteins using electrophoresis
Anode
Buffer chamber
Cathode
Anions Cations
Spotcontaining
proteinmixture
Cellulose acetate strip
+ -
T1
At the end of process, the positions of
proteins are made visible with stains
that bind to proteins .
Before applying current
After
applying
current →
Schematic diagram

Principle of serum protein electrophoresis
❖Principle of Electrophoresis for Separation of serum proteins in clinical
laboratory: most of the serum proteins have PI between 4 and 8 and a net
negative charge at pH 8.6 of buffer( barbiton buffer) .When placed in an
electric field ,they will migrate towards the positively charged electrode
(anode). Albumin carries the largest charge and moves fastest . The globulins
have least net charge and move the least distance .
Point of application
Anode Cathode
 
Migration
Albumin 1 2  

Clinical applications of protein electrophoresis
❖Clinical Applications of electrophoresis in Clinical laboratories :
1. for separation of :
a. Serum Proteins ( albumin moves fastest and  globulins moves the least
distance)
b. Serum Lipoproteins
c. Serum Isoenzymes
d. Hemoglobin
e. separation of other classes of macromolecules.
2. Immunoelectrophoresis : to determine specific classes of Immunoglobulins.
3. Western blot technique :to identify a specific protein e.g. to conform
presence of antibodies to human immunodeficiency virus (HIV).

Separation of Serum proteins by electrophoresis in Clinical
laboratories (diagnostic /therapeutic purpose)
Serumproteins:Albumin,alpha-1globulins,alpha-2globulins,beta-globulins,Gammaglobulins

Separation of Serum lipoproteins by electrophoresis in Clinical
Serum lipoproteins : HDL , VLDL, LDL , chylomicrons

Separation of hemoglobin by electrophoresis in Clinical

Separation of Serum Isoenzymes (CPK and LDH) by electrophoresis in
Clinical laboratories (diagnostic /therapeutic purpose)
SerumCPKIsoenzymes:CK-BB,CK-MB,CK-M SerumLDHIsoenzymes:LDH1,LDH2,LDH3,LDH4,LDH5

Densitometry for Quantitation of proteins
• Densitometry : is a specialized colorimeter designed to scan and
quantitate electrophoretic pattern of proteins.
• Densitometric scanning from cellulose acetate strip converts
characteristic bands to peaks Albumin, -1 globulin, -2 globulin, -
globulin and -globulin.

Technique of Densitometry for Quantitation of proteins :1
❖Technique of Densitometry:
❖The sample is applied to the supporting medium (paper / cellulose acetate/
agar gel / or polyacrylamide gel) and electrophoresis is carried out at
desired constant voltage or constant current in presence of buffer of pH 8.6.
• After completion of electrophoresis, the supporting medium is placed in a
in 7% acetic acid as a fixative to prevent diffusion of separated fractions .
• Separated fractions are visualized by using appropriate stains (bromophenol
blue and Amido schwartz for plasma proteins , Sudan black for
lipoproteins).
• Quantitation of proteins is done by:
a. Densitometry
b. Elution ,followed by colorimetry or spectrophotometry of eluted fractions

Technique of Densitometry for Quantitation of proteins :2
Application of the sample to the supporting medium
ElectrophoresisatdesiredconstantvoltageorconstantcurrentinpresenceofbufferofpH8.6
Fixation of proteins/lipoproteins on the supporting medium in 7% acetic acid so as a to
prevent diffusion of separated fractions
Visualization of separated fractions by using appropriate stains (bromophenol blue and
Amido schwartz for plasma proteins , Sudan black for lipoproteins).
Quantitation of proteins is done by Densitometry /Elution ,followed by colorimetry or
spectrophotometry of eluted fractions

Densitometric scanning for Quantitation of proteins

Principle of Polyacrylamide gel electrophoresis(PAGE) :1
❖Principle of Polyacrylamide gel electrophoresis :separates proteins/ peptide molecule
based on the influence of electric field.
• It is usually a vertical gel electrophoresis .
• Apparatus :columns or slab gels
• Preparation of gel : mixture of the acrylamide (monomers) with N,N’ –methylene bis-
acrylamide (a cross linking agent) in presence of ammonium persulphate (which functions
as an initiator) and N,N, N’,N’ –tetramethylenediamine (TEMED- a free radical catalyst)
• Casting of gel : in a glass tube(sometimes two gels are used on top of one another).
• Spacer gel : loose top gel which concentrates the sample
• Percentage of the acrylamide in gel : varied to suit the type of proteins
Percentage of gel Pore size Application for separation of compounds with
low Large High molecular weight
High Small Low molecular weight

Technique of Polyacrylamide gel electrophoresis

Separationofproteins/peptidesbyslab gelpolyacrylamidegelelectrophoresis(PAGE)
Apparatus for polyacrylamide gel electrophoresis (PAGE) : can be slab or column gel
slab gel PAGE

Stains for Detection of molecules separated by polyacrylamide gel electrophoresis (PAGE)
• Detection : is based on type of molecule
• Quantitation :scanning densitometry ,Elution and spectrophotometric
measurements
Type of Molecule Stains for detection
Proteins Coomassie brilliant blue
Lipoproteins Sudan black
Glycoproteins Periodic acid Schiff( PAS)/lectins

SDS-PAGE for separation of proteins
• Sodium Dodecylsulphate(SDS): is an anionic detergent that disrupts the
secondary ,tertiary and quaternary structures of proteins generating linear
polypeptide chain.
• SDS binds to hydrophobic regions of denatured proteins and masks the
native charge of the proteins .
• Under these conditions , the proteins have constant charge to mass ratio and
electrophoretic mobility is dependent only on the molecular size of the
proteins .
• This technique is used to determine the molecular weight of the protein.

Technique of SDS-PAGE for separation of proteins

Mechanism of SDS-PAGE for separation of proteins

Comparison of Native gel and Gradient Polyacrylamide gel
Criteria Native gel Gradient gel
Polyacrylamide gel without SDS Acrylamide concentration varies from
5% at the top of the gel to 25 % at the
bottom of the gel
Applications Used to analyze proteins in their
native state .Separation of
proteins occurs due to different
electrophoretic mobilities and
sieving effects of the gel
Used to separate proteins of wide
range of molecular weights as well as
proteins of very similar molecular
weight

Applications of Polyacrylamide gel electrophoresis in protein chemistry
❖Applications of Polyacrylamide gel electrophoresis in protein chemistry:
• Separation of proteins/peptides
• Analysis of oligomeric proteins by using solubilizers such as urea ,2-
mercaptoethanol and sodium dodecyl sulphate
• Determination of molecular weight of proteins by SDS-PAGE
❖Other applications of PAGE:
a. DNA sequencing
b. Restriction mapping
c. Separation of nucleic acids

Principle and applications Immuno-electrophoresis
❖Principle of Immuno-electrophoresis : It involves combination of
the principle of electrophoresis and immunological reactions .
Proteins are precipitated at the site of antigen-antibody reaction
and are separated on the basis of electrophoretic patterns.
❖Applications of Immuno-electrophoresis :
1. It is useful for the analysis of complex mixtures of antigens and
antibodies .
2. provides quantitative measurement of proteins –especially
specific classes of immunoglobulins .

TechniqueofImmuno-electrophoresis of serum proteins
Application of human serum ( the mixture of proteins in biological sample) on the gel
Start electrophoresis for separation of proteins
Application of antibody (antihuman serum from rabbit /horse) in a tough parallel to
electrophoretic separation
Diffusion of antibodies in gel
Formation of precipitin bands after contact of antibody with antigen
Identification of protein based on the antibody specificity

Diagrammatic representation of Immuno-electrophoresis of serum proteins
Formation of precipitin
bands/ arc after contact of
antibody with antigen
Trough with antibodies
Electrophoretically separated serum proteins
Diffusion
of
antibodies
in gel

Schematic diagram of Immuno-electrophoresis
Proteins are precipitated at the site of antigen-antibody reaction and
are separated on the basis of electrophoretic patterns.
Precipitin line

Importance of isoelectric pH of proteins
❖isoelectric pH : that pH at which the protein has equal number of positive
and negative charges ( i.e. no net charge). All ionizable groups present in the
proteins influence the pI of the proteins.
❖Proteins at pI exhibit minimum :
a. Buffering capacity
b. Viscosity
c. solubility
❖Proteins tend to aggregate and precipitate at their isoelectric pH . As a result,
they exhibit maximum precipitation and the least solubility.
❖At pH values above the isoelectric pH , they carry a net negative charge and
migrate to anode (+ vely charged electrode) . At pH values below the
isoelectric pH , they carry a net positive charge and migrate to cathode (- vely
charged electrode) . Proteins do not move under electrical field at pI .
❖Application : this property of protein has application in the separation of
protein from mixture by electrophoresis . Hence ,for the electrophoretic
separation of plasma proteins , one has to select a pH different from the
isoelectric pH .

Isoelectric pH of proteins
❖At physiological p H ( 7.4 ) proteins exist as charged molecules assist Donan equilibrium.
Protein PI Charge on proteins at physiological pH( 7.4)
Pepsin 1.1 negative
Casein/egg albumin 4.6 negative
Human albumin 4.7 negative
Urease 5.0 negative
Human Insulin 5.4 negative
Human  globulin 6.6 negative
Human Hemoglobin 6.7 negative
Myoglobin 7.0 negative
Chymotrypsinogen 9.5 Positive
Cytochrome C 10.7 positive
Lysozyme 11.0 Positive

Principle and Technique of isoelectric focusing
❖Principle of Isoelectric focusing ( IEF): is a technique that separates
amphoteric molecules such as Proteins based on the differences in their
isoelectric pH(pI).
❖Technique of isoelectric focusing:
1. Electrophoresis is carried out in the pH gradient created by introduction of
ampholytes into the gel.
2. Proteins are applied on the gel and electricity applied .
3. Each protein moves in the gradient until reaches a pH equal to its
isoelectric pH(pI). At this point, the net charge on the protein is zero and
migration ceases and it forms a band/narrow zone .
4. Since the diffusion is offset by the electrical field , the bonds do not
broaden as in other separation methods.

TechniqueofIsoelectricfocusingtodeterminePIvalueofproteins:1
Set up stable pH gradients in a gel ( using ampholytes)covering the pH range to
include the isoelectric points of the components in a mixture of proteins
Start Electrophoresis for separation of proteins
Migrationoftheproteinmoleculestopositionscorrespondingtotheirisoelectricpoints
Immobilization of proteins at pI and formation of sharp stationary bands
Staining of gel blocks
Identification of proteins based on pI value

Techniqueof Isoelectric focusing to determine PI value of proteins:2
For the proteins in the solution , there is a pH at which the number of anions
formed is exactly equal to the number of cations . This pH is called the
isoelectric pH ( pI ) of that protein and exist as Zwitterion.

Applications of isoelectric focusing (IEF) in Protein chemistry
❖Applications of isoelectric focusing in Protein chemistry :
• Analysis of the microheterogeneity of a protein such as a protein can exist in
mono ,di and tri phosphorylated forms.
• Separation , purification and characterization of proteins.
• IEF is capable of separating proteins that differ in their isoelectric pH(pI) by
only 0.01 of a pH units .
• Capable of resolving serum proteins into 40 bands ( tremendously powerful
method) .

Principle and applications of Isoelectric focusing to determine PI value of proteins
❖Principle of Isoelectric focusing to determine PI value of proteins : is
primarily based on the immobilization of the molecule at isoelectric pH during
electrophoresis .
➢Gel contains a substances called ampholyte which creates a stable pH
gradient in the gel.
➢Since the diffusion is offset by the electrical field , the bands do not broaden
as in other separation method.
❖Applications of Isoelectric focusing :
1. separation of serum proteins (separated to as many as 40 bands)
2. conveniently used for the purification of proteins
3. Tremendously powerful method and is capable of resolving proteins that
differ in pH by as little as 0.01 pH units

Applications of Isoelectric pH of proteins in electrophoretic separation
Application of Isoelectric pH of proteins in electrophoretic separation :
❖At pH 8.6 ( Barbitone buffer ), Albumin (PI =4.7) & Globulin ( PI = 6.7 )carry
negative charge and move towards anode during Protein electrophoresis
based on molecular weight .
Criteria pH < pI pH = pI pH > pI
Charge on amino
acids
Positive charge Neutral Negative charge
Electrophoretic
mobility
Move towards
cathode(cation)
Isoelectric
focusing
Move towards anode
(anion)

Application of Isoelectric pH of proteins in staining of cells
for diagnostic purpose
Type of dye Example Reaction involved in staining
Acidic Eosin Eosin→ H+ + dye
H+ + protein → protein-NH3
+
(protein cation –red colored )
Basic Hematoxylin,
Methylene blue
Hematoxylin/ Methylene blue →
OH
- + dye
OH
- + protein → protein-COO-
(protein anion –blue colored )
Thus, the staining characteristic of a protein is determined by Isoelectric
pH of the concerned protein.

Criteria for Purity of proteins
❖Methods employed to test the Purity or homogeneity of isolated proteins :
Method Criteria for Purity of protein
Solubilitycurve A single sharp break occurs in straight line at saturation point. More than one
breaks in the line or a non-linear curve indicates non-homogeneity of the
sample.
Molecularweight Molecularweightdeterminedbyelectrophoresis,gelfiltration,orultracentrifugation
indicatesthepurityofprotein.
Ultracentrifugation Analyticalultracentrifugationgivesasharpmovingboundarybetweenthepuresolventand
solutecontaininglayer.
Electrophoresis Electrophoreticmobilityandahomogenoussharplevelindicatesthattheproteinispure.
Chromatography Theparticlesofapureproteinemergeoutthechromatographycolumnasonesinglepeak
ofeluent.
Immunoreactivity Theantibodiesraisedagainstapureproteinwillshowonlyonesharpspikeon
immunodiffusionandimmunoelectrophoresis.

Solubilitycurve:criteria for Purity of proteins
Solute
in
solution
Solute added
Saturation point
Homogenous solute
Heterogenous solute
The solute is added repeatedly in increasing quantity to the solvent , the solute in solution rises proportionality
and produces straight line its Solubility curve. When the solution gets saturated, it fails to hold the solute in
solution further.

Analytical techniques for separation or purification of proteins

Analytical techniques for separation or purification of proteins

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