SDS-PAGE

Sodium dodecyl sulphate

polyacrylamide gel
Electrophoresis
Presented by: Azeem Azam

(M.Phil-10)

Hamza Faseeh

(Mphil-02)
 Overview

Part 1 Presented By MPhil 2 Part 2 Presented by MPhil 10

• Introduction • Assembling gel casting apparatus

• History • Gel preparation
• Principle of SDS-PAGE • Gel casting
• Sample loading
• What are Proteins?
• Running gel
• Protein Sample preparation
• Visualization of proteins
• Instrumentation and Chemical • Applications
components of system
• Drawback
 INTRODUCTION
Gel electrophoresisis a widely used technique for the analysis
of nucleic acids and proteins.

Gel electrophoresis is a procedure that separates molecules

on the basis of their movement through a gel under the
influence of an electrical field.
SDS(sodium dodecyl sulphate)–Polyacrylamide gel
Electrophoresis is routinely used for the estimation of protein
subunits according to their molecular weights.
What is Polyacrylamide Gel Electrophoresis (PAGE)?

Polyacrylamide gel electrophoresis (PAGE) is a

technique widely used in biochemistry, forensic
chemistry, genetics, molecular biology and
biotechnology to,
‘separate biological macromolecules, mainly proteins
or nucleic acids, according to their electrophoretic
mobility’.
History..

SDS-PAGE is a discontinuous electrophoretic

system developed by U.K. Laemmli

which is commonly used as a method to separate

proteins with molecular masses between 5 and 250
KDa
Principle of SDS-PAGE

SDS-PAGE is used to separate proteins in an electric field based on

》 Size(molecular
weight –MW)

》 Rate of migration • Proteins migrate at different rates

through the gel depending upon the size .
What are proteins?
A protein consists of one or more polypeptides ,held together by any
of a number of molecular interactions often including covalent
bonds. Such interactions result in several levels of organization,
which we call primary, secondary, tertiary, and quaternary structures.

A polypeptide is a macromolecule consisting of a nonbranching

sequence of amino acids, each connected to the next by a single
peptide bond.
 Continued..
Native protein structure exhibits disulfide bonds and non covalent
interactions that confers its 3D structure(i.e folding).

Non covalent interactions include

》 Hydrogen and ionic bond interactions(polar hydrophilic).

》 Hydrophobic interactions(non-polar).
Continued..
Generally, proteins are amphoteric
molecules that possess both positive as well
as negative charges within the same
molecule. Therefore, a uniform negative
charge is given to protein molecules in order
to move them in a single direction during
electrophoresis.
 Protein sample preparation:

• SDS
• Buffer tris HCl,pH 6.8
Component • Beta mercaptoethanol(Reducing agent);
s of sample reduce disulfide bonds
• Glycerol:provide viscosity; sink sample in wells
buffer are • Bromophenol blue dye: allows tracking of gel’s
progress.
 Sodium dodecyl sulfate(SDS)

Sodium Dodecyl Sulphate (SDS)dissociates proteins into their individual

polypeptide subunits.Hydrophobic acyl chain of sodium dodecyl sulfate binds to
a proteins amino acid residues and it gives a uniform negative charge(from
sulfate)along each denatured polypeptide.

Number of negative charge is proportional to the protein size(constant mass to

charge ratio).
 Beta-mercaptoethanol

Beta-
mercaptoethan
ol further
denatures by
reducing
disulphide
linkages thus
overcoming
tertiary protein
folds

• After addition of the sample buffer components the mixture is heated for 5
mins at 95 celsius to denature the proteins. Now the sample is ready.
 The Gel (matrix)
The gel (matrix) itself is composed
of either agarose or
polyacrylamide.
Polyacrylamide is a cross-linked
polymer of acrylamide.

* Acrylamide is a potent neurotoxin.

Polyacrylamide gels:
Polyacrylamide is used to form gel, a matrix of pore which allow
the molecules to migrate at different rates.

Have smaller pores size than agarose therefore high degree of

resolving power.

The size of pores is determined by the concentration of

acrylamide.

The higher the concentration of acrylamide, the smaller the pore

size.
Why polyacrylamide is used for a
gel?
Chemically inert

Electrically
neutral

Hydrophilic

Transparent for
optical
detection
Components of Polacrylamide gel
Deionized water
TEMED
SDS
Acrylamide
Bisacrylamide
Ammonium persulphate
• Tris hcl buffer
Note:
The exact amounts of these components vary according to the type of gel (either
stacking or resolving)
Instruments required
Electrophoresis tank

DC power supply

Glass plates

Casting frame

Casting stand

Spacers

Combs
 Chemicals required

- Gel (polyacrylamide)

- Buffer

- Proteins

- Nucleic acids
 Azeem Azam
• Roll no. 10
 Assembling the Apparatus
Assemble the components that you will need for casting the gel:
1- plates (tall and short)
2-casting frame
3-casting stand

Place the short plate in front of the taller plate .

Fix these plates with spacer having 1 mm distance between them.
Now place these plates in casting frame.
Now this frame having plates is to be fixed in casting stand.

Now put comb between the plates from the top. Mark a line 1 cm below the comb.
This line is used to make the boundaries of the separating and stacking gels.
Remove the comb after marking. Now ,we have to check the leakage of plates,by pouring water in it.
If there is no leakage , remove the water using filter paper.
Glass plate set
Casting frame Casting stand
Combs
 Casting the gel
• There are two types of gels used in this technique, Namely
resolving and stacking gel, their composition is as follows;
Polymerization of Acrylamide and Bis-Acrylamide

Acrylamide and Bisacrylamide, both are very important for proper

polymerization.

Acrylamide forms linear polymers whereas Bisacrylamide cross links

these linear polymers.

TEMED accelerates the rate of formation of free radicals from

persulfate and these in turn catalyse polymerization. The persulfate
free radicals convert acrylamide monomers to free radicals which react
with inactivated monomers to begin the polymerization chain reaction.
Mechanism of Polyacrylamide Gel
formation
 Casting the gel
1.Resolving gel preparation(10 to 15% acrylamide)
• Resolving gel is small pore polyacrylamide gel
• It known as running gel or separating gel
• Allows to separate proteins based on their molecular weight
.
• REAGENTS:
- Deionized water
- 30% acrylamide, Bis acrylamide
- 1.5M Tris HCl ,40% SDS, pH 8.8
- 10%ammonium persulfate(catalyst),
- TEMED(catalyst)
Pouring the gel
• Using plastic pipette, the space between two glass
plates is filled with resolving gel solution Uptil the
marked line is reached.
• Bubbles can be romoved using iso- propanol.
• Allow the gel to polymerize.
• After polymerization reomove isoprpanol using filter
paper.
2. Stacking gel Preparation(About 2 to 5% acrylamide)

Stacking gel is a polyacrylamide gel

• Casted over the resolving gel
• Needed to concentrate all the proteins in one band so they will start migrating in
running gel at same time
• REAGENTS :
- Deionized water
- 30% acrylamide Bis acrylamide ,0.4% SDS,
- 0.5 M Tris HCl, pH 6.8
- 10% Ammonium persulfate (Catalyst)
- TEMED(Catalyst)
 How stacking gel
works
• Glycine present in buffer is a zwitter ion(a substance that can
change its charge depending on pH)
• It has negative charge at pH far above pH 5.97,when it enters
the stacking gel (pH 6.8) it is predominantly neutral,
• And trails behind the Proteins, while the chloride ions (being
very negative in charge) move ahead of the proteins.
• Thus concentrating the proteins into one band before entering
the separating gel.
How stacking gel works

• Glycine moves slowly (Being less negative) while

chloride ions move quickly (Being very
electronegative),
• While the proteins move in between them (being
intermediate in charge) forming a thin and compact
layer
• Before entering the stacking gel.
Assembling the gel
• To assemble,Take out the gel plates from the casting frame
and clamp them in to another stand.
• When the plates are secured,place them in the cassette and
then lock.
• Place the cassette in the gel running tank.
• Fill the inner chamber of tank with buffer(Now it is easy to
remove comb since it is lubricated)
• Remove the comb carefully (without breaking wells).
• (Now gel is ready to load Samples).
Loading of sample
Insert loading tip to a few mm
from well bottom and deliver the
samples into the well
Running the gel
• Cover the tank with lid and connect the electrophoresis tank
to the power supply.
• Allow the samples to run at 180 V until Dye front reaches
the bottom of gel.
Staining the gel
• Remove the gel from the plates
• Put the gel in stain
• The stain solution will make protein bands visible
• The protein band will appear under the influence of ultraviolet
radiations
Destaining the gel
• Destain the gel until the Bands are properly seen.
• Determine the approximate molecular weight of visualized
protein bands by comparing them with molecular weight
ladders(markers).
Overview of loading,running and staining
sample
Safety precautions
• Keep container tightly closed
• Keep ignition sources well clear of TEMED vapour, mostly 3
meter is advisable
• Don’t inhale TEMED
• Remove the combs gently
• Insert protein sample carefully in wells without disturbing the
wells
• Comparison of polypeptide chain of different protein samples
• Analysis of the number and size of polypeptide chain
Applications of SDS-PAGE
• SDS-PAGE is used mainly for the following purposes:
• Determine purity of protein samples
• Determine molecular weight of protein
• Identify disulfide bonds between protein
• quantify proteins
• Monitoring protein integrity
• Peptide mapping
• Post electrophoresis applications such as western blotting
Drawback
• The drawback of this technique is that proteins are denatured.
• If protein strucure is required to be maintained, native PAGE is used.