PROTEIN ANALYSIS

PROTEIN ANALYSIS;Two dimensional separation of total
cellular proteins
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SUBMITTED BY:
VIPIN E.V
CLASS.NO:373
I-M.Sc. APPLIED MICROBIOLOGY

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 SDS-PAGE (SDS-polyacrylamide gel
electrophoresis)
 Isoelectric focussing
 High-performance liquid
chromatography (HPLC)
 Thin-layer chromatography (TLC)
 Two-dimensional (2-D) gel
electrophoresis

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SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
 separates proteins mainly
on the basis of molecular weight

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Isoelectric focussing
 Separates proteins on the basis of their balance of acidic
(negatively charged) vs. basic (positively charged) amino acid residues,
which
determines a property known as the protein’s isoelectric point [pI],
 proteins with very similar pI values but very different sizes can run
at the same position.
High-performance liquid chromatography (HPLC)
 used to separate and to purify proteins/peptides based on size,
charge or overall hydrophobicity.

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Thin-layer chromatography (TLC)
 used to separate out peptides (e.g., derived from proteolytic digestion of a
protein) based on similar properties.
Two-dimensional (2-D) gel electrophoresis
 Two-dimensional (2-D) gel electrophoresis is a powerful gel-based
method commonly used for ‘global’ analysis of complex samples
In this technique the protein is run first in a narrow (often
tube-shaped) isoelectric focussing gel, which as already noted
separates proteins on the basis of their isoelectric points (acid vs.
basic character).

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• The mixture of proteins is loaded onto a gel that has a gradient of increasing pH.
• An electric field is applied and the proteins move along the pH gradient until they
reach the point at which their charges are neutralized.
• At this point, each band in the gel contains several different proteins with the
same (or very similar) isoelectric point. The tube gel is removed from its tube
and exposed to SDS to denature the proteins
• It is then placed on a slab of polyacrylamide gel and traditional SDS-PAGE is run
in the second dimension to separate the proteins by size. After staining,
the result of 2D-P AGE is a square with small scattered dots representing
individual proteins
• The first step in separating large numbers of proteins in two dimensions is to
separate them according to their inherent charge.

INTRODUCTION
GEL ELECTROPHORESIS
PRINCIPLE
BASIC CONCEPT
SUPPORT MEDIA
BUFFERS
APPARATUS
AGE
PAGE
SDS PAGE
APPLICATIONS
CONCLUSIONS
BIBLIOGRAPHY
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 Father of Electrophoresis - Arne W.K. Tiselius
(Swedish physical biochemist , 1902-1971)
 The Nobel Prize in Chemistry 1948 for the discovery of proteins in blood serum
and for the development of electrophoresis as a technique for studying proteins.
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Gel electrophoresis is one of the most basic
important tools used by the molecular
biologists in the study of DNA or its fragments.
Gel electrophoresis technique is used-
 To study the purity and intactness of DNA;
 To separate and identify the fragments;
 To analyze and characterize recombinant
DNA molecules.
 To determine molecular weight of DNA
fragments by running standard markers in
parallel.
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When a voltage is applied across the electrodes, it generates a potential gradient E,
E=v/d
E=potential difference between the electrodes V=applied voltage, D=distance between
the electrodes
When this potential E is applied, the force on a molecule is Eq newtons where q is
charge (in coloumbs) of molecule.
The velocity v of a charged molecule in an electric field is v=Eq/f where
F=frictional coefficient.
The term electrophoretic mobility μ= v/E is the ratio of velocity to field strength.
In electrophoresis, separation is based on charges or molecular size. The current in the
solution between electrode is conducted by buffer ions. Ohm’s law gives relation
between current, voltage and resistance
V/I=R Increase in voltage increases the electrophoretic mobility.
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Most of the power generated is dissipated as heat. Heating of the
electrophoretic medium has the following effects:
 Increased rate of diffusion of sample and buffer ions leading to broadening
of the bands
 Temp sensitive protein /enzyme samples tends to denature
 Decrease in buffer viscosity, increase in mobility.
 Smiling effect (unequal heating of gel) temp of centre of gel is higher than
the rest of the gel, reduction in viscosity, faster is the migration.
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Gel electrophoresis is a method that separates macro molecules-either nucleic
acids or proteins-on the basis of size, electric charge, molecular mass and other
physical properties.
Migration of ions in an electric field at a definite pH is called as electrophoresis.
Gels are used for 2 reasons:
Gels suppress convection current produced by small temperature gradients, a
requirement for effective separation and
Gels resolve as molecular sieves that enhances the separation.
Migration of a molecule is inversely proportional to its frictional coefficient while
frictional coefficient is dependent on size and shape of the molecule. But DNA
molecule can assume different conformations (shapes) and this fact complicates
electrophoretic analysis of DNA. ( plasmids migrate considerably faster than open
circles (OC) and linear plasmid (L) migrates just ahead of the open circle.)
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Many important biological molecules such as amino acids, peptides, proteins,
nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH,
exist in solution as electrically charged species either as cations (+)or anions(-).
Depending on the nature of the net charge, the charged particles will migrate either to
the cathode or to the anode.
The rate of this electrophoretic migration or mobility depends on the pH of the
medium, strength of the electric field, magnitude of the net charge on the molecule and
the size of the molecule.
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It is important that the support media is electrically neutral. Presence of charge group
may cause:-
Migration retardation
The flow of water toward one or the other electrode so called ‘Electroendosmosis
(EEO)’, which decrease resolution of the separation
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AGAROSE GELS
For the separation of :
(1) large protein or protein complex
(2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose in a gel
(normally in the rank of 0.4-4%)
OH
O
OH
CH2OH O
O OH
O
O
O
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CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CH-
-CH2-CH-CH2-CH-CH2-CH-
CO
NH
CH2
NH
CO
CO
NH
CH2
NH
CO
CO
NH2
NH2
CO
n
n
POLY ACRYLAMIDE GELS
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 Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
 High ionic strength of buffer
 produce sharper band
 Commonly used buffer
 Barbital buffer & Tris-EDTA for protein
 Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
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It includes:
 A power pack -to supply uninterrupted
constant current.
 Electrophoresis unit:
 Gel casting tray -to cast the Agarose gel.
 Plastic combs -to form loading wells for
sample in the gel.
 Electrophoresis tank -to pour buffer, (usually
TAE/TBE) provide ions to support
conductivity.
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Agarose is a linear polysaccharide isolated from certain sea weeds.
Formation-dry agarose boiled in aq. Buffer to form clear solution, it is then poured
& allowed to cool at room temp to form a rigid gel.
Gelling properties are due to the inter & intra molecular H bonding within &
between the long agarose chains.
polymers during gel formation
Low concentrations of agarose–forms gels
with large pores.
It is sold in different purity grades based on
the sulphate concentration.
Used– electrophoresis of proteins & nucleic
acids.
Advantage-can be reliquified by heating to
65̊c(DNA samples separated in the gel can be
recovered).
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AGE
Scheme of the method
A photograph of a gel after electrophoresis
showing the DNA fragments as bright
bands. DNA binds to a dye that fluoresces
orange under ultraviolet light.
http://en.wikipedia.org/wiki/Gel_electrophoresis23

HORIZONTAL AGAROSE GEL ELECTROPHORESIS
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Detect bands by staining during or after electrophoresis
Ethidium bromide: for double-stranded DNA
SyBr green or SyBr gold: for single- or double-stranded DNA or for RNA.
Silver stain: more sensitive for single- or double-stranded DNA or for RNA and
proteins.
DETECTIONOF BANDS
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APPLICATIONS
Determination of DNA sequences.
Southern and Northern blotting.
Restriction mapping of DNA.
Determination of subunit of protein.
Determination of molecular weight of protein.
Measure distance migrated for selected
unknown proteins on gel
Determine size of unknowns from the graph
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Poly acrylamide gels are the supporting media of choice for electrophoresis because
they are chemically inert.
Formed from the polymerization of acrylamide monomer in the presence of N, N’-
methylene bis acrylamide.
Polymerization is initiated by the addition of ammonium per sulphate (APS) &base
tetra methylene diamine (TEMED). TEMED catalyses the decomposition of
persulphate ion to give a free radical.
O2 removes free radicals therefore the solutions are placed under vacuum to remove
loosely dissolved O2 before use.
Pore size-varied by changing the concentration of acrylamide & bis-acrylamide.
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Courtesy
by:http://www.google.co.in/imgres?imgurl=
http://www.google.co.in/page/
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Polymerization should take place under an inert atmosphere since oxygen can act as a
free radical trap.
The polymerization is temperature dependent : to prevent incomplete polymerization
the temperature should be maintained above 20⁰c.
To minimize oxygen absorption gels are usually polymerized in vertical casting
chambers.
The gel is divided into two areas: resolving and stacking gel.
The resolving gel with small pores of pH 8.8 , the stacking gel with large pores of pH
6.8. (to concentrate the protein sample before it enters resolving gel).
Non-denaturing polyacrylamide gel
electrophoresis of proteins from
induced E. coli.
http://www.biomedcentral.com/1471-
2091/2/13/figure/F5
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In this method of electrophoresis, the polyacrylamide gel contains 0.1% SDS, and the
proteins are treated with an excess of SDS ( sodium dodecyl Sulphate), which is
anionic (negatively charged) detergent.
SDS has the following effect on protein molecules:
(1) all hydrogen bonds are broken,
(2) all hydrophobic interactions are cancelled,
(3) aggregation of the proteins is prevented,
(4) individual charge differences of the proteins are masked, and
(5) the polypeptides become unfolded (removal of tertiary structure)
***However, SDS does not affect disulphide bonds and hence β-mercaptoethanol is
used.(samples are boiled for 5min in buffer sample containing both SDS and β-
mercaptoethanol )
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http://upload.wikimedia.org/wikipedia/commons/thumb/4/46/ 32

The binding of the SDS to the protein molecule imparts a –ve charge to the protein
and hence here the proteins are separated only on the basis of its net molecular mass
(size).
Stacking gel (large pore size) concentrates the protein sample into a sharp band
before it enters main sep. gel-achieved by the differences in ionic strength & pH
between the electrophoresis buffer & the stacking gel.
The hydrocarbon chains of
the SDS can get linked to the
hydrophobic regions of the
interior in the interior of
proteins(globular) , leaving
the ionized sulphate groups,
jetting out into the solvent
medium.
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Sample buffer contains an ionisable tracking dye (bromophenol blue-monitors
electrophoretic run)&sucrose/glycerol(gives density to the sample).
Application of electric field makes smaller proteins move faster in the gel than the
larger ones which are retarded by frictional resistance due to the sieving effect of the
gels.
After the run reaches ¾ of the gel, it
is removed from between the glass
plates & shaken in a stain solution
(coomassie brilliant blue) for few hrs.
& then washed in destain solution
overnight (removes unbound
background dye).
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Samples- whole tissue/cell culture.
Solid tissues-broken down-using blender (larger mol.) and using a
homogenizer(smaller mol.) or by sonicator.
Filtration and centrifugation – used to separate different cell compartments and
organelles.
Proteins to be analyzed – mixed with SDS which denatures the secondary and non-
disulfide linked tertiary structures and applies a negative charge to each protein in
proportion of its mass – required a heating procedure.
Tracking dye- added to the protein soln.

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Acrylamide + bisacrylamide +SDS + Tris-Cl buffer + adjust pH.
Soln. degassed under vacuum - prevent bubbles during polymerization.
Separating/resolving gel is more basic than the stacking/loading gel.
Gels polymerized in gel caster.
1st separating gel poured and allowed to polymerize.
Isopropanol added (thin layer) to form a smooth surface.
Loading gel poured + comb placed to create wells.

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Anode buffer (Tris-Cl , D deionized wa + pH higher than cathode buffer) , cathode
buffers (SDS +Tris + Tricine + D deionized water) prepared.
Cathode buffer – covering the gel in the –ve electrode chamber.
Anode buffer in the lower +ve electrode chamber.
Denatured protein samples added to the wells.
Apparatus kept for electrophoresis.
An electric field is applied across the gel and the –vely charged proteins migrate
towards the +ve electrode.
Depending on their size each protein will move differently through the gel matrix
(larger molecules encounter more resistance).

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Proteins separated by SDS
PAGE method
http://jpkc.scu.edu.cn/ywwy/zbs
w(E)/pic/ech3-9.jpg
Uses-for analyzing protein mixtures
qualitatively.
-Useful for monitoring protein purification.
-Determine molecular mass of proteins.
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Staining with Coomasie blue
STAIN DETECTION LIMIT
Ponceau stain 1-2 mg
Amido Black 1-2 mg
Coomassie Blue 1.5 mg
India Ink 100 ng
Silver Stain 10 ng
Colloidal Gold 3 ng
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Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in
restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting.
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to
Northern transfer.
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 By conducting a study on Gel electrophoresis and the types of gels (agarose and
polyacrylamide gels) used for this technique, we understand the various principles
and theory behind performing this technique.
 We also learnt its importance in the study of the genetic materials (especially
DNA and its fragments), which are the basic elements of all life forms.
RNA and other important proteins are also studied with the help of gel
electrophoresis technique.
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Books
 Narayanan . P. ; Essentials of Biophysics; 2nd edition ; New Age International (P)ltd.; New-Delhi;
pg. no: 186-189
 De Robertis . E.D.P., De Robertis . E.M.F; Cell and molecular biology; 8th edition; Info-Med ltd;
pg.no:50-51..
 Jain.J.L, Jain Sunjay, Jain Nitin; Fundamentals Of Biochemistry; 8th edition; S.Chand and
company ltd.; New-Delhi; 1040-1043
Journals
 Tonge, R, et al , ‘Validation and development of fluorescence two-dimensional differential gel
electrophoresis proteomics technology’ , Journal of Proteonomics, volume 1, Viewed on 12 Nov
2013,http://onlinelibrary.wiley.com/doi/10.1002/1615-9861(200103)1:3%3C377::AID-
PROT377%3E3.0.CO;2-6/abstract
Websites
 http://ai.stanford.edu/~serafim/CS262_2007/notes/lecture10.pdf
 http://en.wikipedia.org/wiki/Gel_electrophoresis>
 http://m.wisegeek.org/what-is-agarose -gel.htm
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