-Introduction
-Process technique
-Uses and future Prospect
Electrophoresis…
Electrophoresis is a
laboratory technique for
separating molecules
based on their charge
The gel (matrix)

The gel (matrix) itself is composed of either
agarose or polyacrylamide.

Polyacrylamide is a cross-linked polymer of
acrylamide.
Polyacrylamide gels…

Have smaller pores than agarose, therefore high
degree of resolving power.

Can separate DNA fragments which range in size
from 10-500 bp.

DNA fragments which differ in size by one
nucleotide can be separated from each other.

Polyacrylamide gel electrophoresis is mostly used
to separate protein molecules.
Protein Electrophoresis
 Proteins,
unlike DNA, do not have a
constant size to charge ratio
 In
an electric field, some will move to the
positive and some to the negative pole, and
some will not move because they are
neutral.
SDS-PAGE (PolyAcrylamide Gel
Electrophoresis)…

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in biochemistry,
forensics, genetics and molecular biology:

to separate proteins according to their electrophoretic
mobility (a function of length of polypeptide chain or
molecular weight).

to separate proteins according to their size, and no other
physical feature.
SDS…

SDS (the detergent soap) breaks up hydrophobic areas and
coats proteins with negative charges thus overwhelming
positive charges in the protein.

The detergent binds to hydrophobic regions in a constant
ratio of about 1.4 g of SDS per gram of protein.

Therefore, if a cell is incubated with SDS, the membranes
will be dissolved, all the proteins will be solubalized by the
detergent and all the proteins will be covered with many
negative charges.
PAGE…
If the proteins are denatured and put into an electric field
(only), they will all move towards the positive pole at the
same rate, with no separation by size.

However, if the proteins are put into an environment that
will allow different sized proteins to move at different
rates.

The environment is polyacrylamide.

the entire process is called polyacrylamide gel
electrophoresis (PAGE).
Basma Sarfraz

Samples may be any material containing proteins

Solid tissues:
Broken down mechanically using a
1.
Blender (for larger sample volumes)
2.
Homogenizer (smaller volumes)
3.
Sonicator
4.
Cycling of high pressure.

Soft tissues or cells:
Broken down by a combination of biochemical and mechanical techniques as
1. Filtration
2. Centrifugation
3. Electrophoresis

Materials Required For PAGE:

Protein samples .

Molecular weight markers.

Acrylamide solutions (for resolving & stacking gels).

Isopropanol / distilled water .

Gel loading buffer.

Running buffer.

Staining, destaining solutions.

The equipment and supplies necessary for conducting SDS-PAGE includes:

An electrophoresis chamber and power supply.

Glass plates(a short and a top plate).

Casting frame .

Casting stand.

Combs .
It possesses several electrophoretically desirable features that make it a versatile medium. It is
a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, can also
withstand high voltage gradients, is amenable to various staining and destaining procedures, and
can be digested to extract separated fractions or dried for autoradiography and permanent
recording and can be prepared with a wide range of average pore sizes.

Gels typically consist of
1.
Acrylamide
2.
Bisacrylamide
3.
SDS
4.
Buffer with an adjusted pH
5.
Ammonium persulfate
6.
TEMED
Recipe for a traditional 10%
mini gel for SDS-PAGE:
1.
2.
3.
4.
5.
6.
7.
7.5mL 40% acrylamide solution
3.9mL 1% bisacrylamide solution
7.5mL 1.5 M Tris•HCl, pH 8.7
Add water to 30mL
0.3mL 10% APS
0.3mL 10% SDS
0.03mL TEMED

Regardless of the system, preparation requires casting two different layers of acrylamide between glass
plates.

The upper layer (stacking gel) includes the sample wells. It is designed to sweep up proteins in a sample
between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when
they reach the separating gel.

The lower layer (separating, or resolving, gel) is responsible for actually separating polypeptides by size.

The ionic strength discontinuity between the loose stacking gel and the hard running gel leads to a
voltage discontinuity as current is applied. The goal of these gels is to maximize resolution of protein
molecules by reducing and concentrating the sample to an ultrathin zone (1-100 nm) at the stacking
gel:running gel boundary. The protein sample is applied in a well within the stacking gel

The protein sample contains glycerol or sucrose so that it can be overlaid with a running buffer.
Resolving gel 10% Stacking gel 5%
dH20
30% acrylamide mix
1.5M Tris pH8.8
10% SDS
10% ammonium persulfate
TEMED
4.0 ml
3.3 ml
2.5 ml
0.1 ml
0.1 ml
0.004ml
5.65ml
1.65ml
pH 6.8

unreactive and not modify with most proteins.

Significance:

Stabilizes the pH value to the desired value

Electrophoretic mobility- ions
Gel loading buffer:

Common buffers in SDS-PAGE include Tris, Bis-Tris, or imidazole.
To make 10 mL of 4X stock:
2.0 ml 1M Tris-HCl pH 6.8.
0.8 g SDS.
4.0 ml 100% glycerol.
0.4 ml 14.7 M β-mercaptoethanol.
1.0 ml 0.5 M EDTA.
8 mg bromophenol Blue.

Electroporesis separation depends upon:
1.
Protein’s size ( molecular weight)
2.
Gel pores (density)
3.
Voltage applied
Exceptions:
Glycoproteins
• Finding of approximate
molecular weight…
By running molecular weight
size markers of known molecular
weight in a separate lane in the
gel.

Staining:
The gel may be stained most commonly with Coomassie Brilliant Blue R-250 or Silver
stain.
Western
different proteins will appear
as distinct bands within the gel
blotting
Allowing visualization of the separated proteins, or processed further.

Result:
Different proteins will appear as distinct bands within the gel. Alternatively, if
the proteins are radioactive, the protein bands can be detected by
autoradiography.

Stained gels can be dried and preserved, photographed or scanned with a
recording densitometer to measure the intensity of the color in each protein
band.
Weigh 0.25g of
Coomassie Brilliant
Blue R250 in a
beaker. Add 90 ml
methanol:water
(1:1 v/v) and 10ml
of acetic acid.
SDS-PAGE is usually the first choice as an assay of protein purity due to its reliability and ease.
1.
Presence of SDS
2.
Denaturing step
3.
Aberrant migration of some proteins
4.
Differently staining interferes with quantification.
preparative
technique for the
purification of
proteins-QPNC-PAGE
Uses and Future
prospective of PAGE…
komal jamim shehzadi
WHAT IS SPECIAL ABOUT SDS PAGE

SDS is a negatively charged detergent

Disrupt secondary and tertiary structures by breaking hydrogen bonds and
unfolding proteins.

MASKS charge on protein so that all protein act the same as regards charge.

Prevents protein aggregation.

Prevents protein shape from influencing gel run.
Sodium Dodecylsulphate
Now we are ready to focus on the second half - PAGE.
SDS and Proteins
SDS
Protein
SDS and Proteins

SDS nonpolar chains arrange themselves on proteins
and destroy secondary tertiary and quarternary
structrure

So much SDS binds to proteins that the negative
charge on the SDS drowns out any net charge on
protein side chains
In the presence of SDS all proteins have uniform
shape and charge per unit length

Uses of SDS page…

Determine protein size

Identify protein

Determine sample purity

Identify existence of disulphide bond

Quantify amounts of protein

Recycle back to the PM

Recycle back to the TGN

Western transfer always begin with SDS page
Cont…

Antibodies can be produced from protein excised from
gel.

Sometimes can reconstitute proteins

Mass spectrometry and proteomic studies

Measuring Molecular Weight with SDS-PAGE

Peptide mapping

Protein concentration determination

Protein crystallization

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera
from lepromatous patients and infected armadillos.

A K Chakrabarty, M A Maire, and P H Lambert

Copyright and License information ►

This article has been cited by other articles in PMC.

Abstract

Studies have been conducted to characterize M. leprae bacilli derived from infected
armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDSPAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a
strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated
bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react
immunologically with sera from the lepromatous patients, infected armadillo sera and
other experimental mycobacterial antisera. It was observed that a majority of M. leprae
proteins contained antigenic determinants also present on proteins of BCG. In addition,
only two specific antigen bands of 33KD and 12KD were conspicuously detected by the
patients' sera and the infected armadillo sera. These substances were further identified
as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or
alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat
germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD
glycoprotein antigens were found to lose their antigenicity after pepsin treatment and
can be considered as glycoproteins. Further, radiolabelling experiments showed that
12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein
failed to be similarly radiolabelled. It is suggested that these protein antigens have M.
leprae specific determinants on a cross-reacting component.