I am HAFIZ M WASEEM FROM mailsi vehari
BSc in science college Multan Pakistan
MSC university of education Lahore Pakistan
i love Pakistan and my teachers
3. Introduction
What is SDS-PAGE?
Separation of macromolecules on the basis of
their electro-phoretic mobility is
called electrophoresis
The techniques cannot be used to determine
molecular weight of biological molecules
because the mobility of a substance in the gel
depends on both charge and size
So, there is a need to denature the proteins with
some detergent like SDS so that they lose their
secondary, tertiary or quaternary structures and
become linear
4. Introduction
This method is called sodium dodecyl
sulfate polyacrylamide gel
electrophoresis (SDS-PAGE)
The method is also called Laemmli
method after Laemmli who was the first
to publish a paper employing SDS-PAGE
A polypeptide chain binds with SDS in
proportion to its relative molecular mass
This results in fractionation by
approximate size during electrophoresis
5. Introduction
Negative charges on SDS destroy most of the
complex structure of proteins
These molecules are strongly attracted
toward an anode in an electric field
Polyacrylamide gels restrain larger molecules
from migrating as fast as the smaller
molecules
SDS-PAGE is the most widely used analytical
method in biochemistry and molecular
biology
Characterization of proteins is also possible
7. Principle of SDS-PAGE
SDS denatures the proteins by binding to
hydrophobic regions
Non-covalent bonds are disrupted and the
proteins acquire a net negative charge
A Concurrent treatment with a disulfide
reducing agent such as β-mercaptoethanol or
DTT (dithiothreitol) also denature the proteins
by reducing disulfide linkages
Proteins samples get uniform structure and
charge
A suitabe dye is used to monitor electrophoresis
8. Separation depends on their molecular weight
only
Small proteins migrate faster than the larger
ones through the gel matrix under the influence
of the applied electric field
Number of SDS molecules that bind is
proportional to the size of the protein
So, the SDS-treated proteins get similar charge-
to-mass ratios and similar shapes move towards
anode and separate only according to their
molecular weight
Principle of SDS-PAGE
10. Sample containing proteins or nucleic acids is
prepared by using
Homogenizer
Sonicator
Filtration & centrifugation
It is mixed with some suitable denaturant like
SDS for proteins
Proteins are also heated with a reducing agent
like Beta-mercaptoethanol which denatures the
proteins by reducing disulfide linkages thus
breaking the complex structures
Procedure of SDS-PAGE
Sample Preparation
11. SDS-PAGE gels consist of separating and stacking gels
For separating gel prepare gel solution (desired %age)
and pour it into the gap between the glass plates
It is degassed under a vacuum or butanol is added to
prevent the formation of air bubbles during
polymerization
APS and TEMED are added to initiate polymerization
Wash top of the gel for several times to remove
acrylamide that is unpolymerized
Procedure of SDS-PAGE
Preparing the gel
12. Stacking gel (5%) is poured on top of the separating gel
and a comb is inserted to make wells
After polymerization the comb is removed AZis ready
for electrophoresis
Acrylamide concentration may range from 5% to 25%
Lower percentage gels are better for resolving very
high molecular weight molecules
While higher percentages are needed to resolve smaller
proteins
Finally the gel is fixed in the electrophoresis chamber
Procedure of SDS-PAGE
Preparing the gel
13. Samples are mixed with loading buffer and are
loaded in the wells
The ladder is loaded in the first well
Various buffer systems are used in PAGE
depending on the nature of the samples
Generally the gel is run for 1 hour at 120V voltage
for 12% separating gel
Negatively charged proteins or nucleic acids
migrate towards the anode
Smaller molecules move faster than the larger
ones
Procedure of SDS-PAGE
Electrophoresis
14. After electrophoresis, the gel is stained
with dyes like Coomassie Blue
Different Proteins are stained as distinct
bands in the gel
Different proteins are stained differently
with the stains
Detergents like SDS are used to separate
the folded proteins
A suitable marker is used to
estimate molecular mass of the unknown
proteins
Procedure of SDS-PAGE
Staining & Visualization
16. A number of factors affect the rate of
electrophoresis like
Concentration of gels
Size of molecules being electrophoresed
Voltage used
Time
Ionic strength of buffers
Dyes such as ethidium bromide used during
electrophoresis
Characterization of proteins by Western blotting
by transferring to a membrane
Factors affecting SDS-P AGE
18. SDS-PAGE has a number of applications which
include
• Measuring molecular weights of different bio-
molecules
• Estimation of purity of the proteins
• Quantification of proteins
• Analysis of number and size of proteins
subunits
• Characterization of proteins by Western
blotting
• Staining of proteins
Applications of SDS-PAGE