I AM HAFIZ MUHAMMAD WASEEM from mailsi vehari BSc from science college Multan MSC university of education Lahore i love Pakistan and my teachers

1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE
Micrometry

2. Advanced Analytical Techniques
UNIVESITY OF EDUCATION
LAHORE PAKISTAN

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

6. Advanced Analytical Techniques
SDS-Polyacrylamide
Gel Electrophoresis-
Principle

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

9. Advanced Analytical Techniques

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

15. Advanced Analytical Techniques

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

17. Advanced Analytical Techniques

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