SDS-PAGE

Standard test that used to determine the charged molecules, mainly proteins and nucleic acids.

Widely used in biochemistry, forensics, genetics and molecular biology.

Laemmli system of SDS-PAGE was first introduced in 1970s

1. Sodium Dodecylsulfate Sodium Dodecylsulfate
Polyacrylamide Gel Polyacrylamide Gel
Electrophoresis(SDS-PAGE)Electrophoresis(SDS-PAGE)
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. Overview Overview
a.Introduction
b.Principle of Sds-page
c.Gel preparation
d.Process of Sds-page
e.Visualization of protein
f.Applications
g.Advantages and disadvantages
h.Conclusion
i.References

3. IntroductionIntroduction
 Standard test that used to determine
the charged molecules, mainly proteins
and nucleic acids.
 Widely used in biochemistry,
forensics, genetics and molecular
biology.
 Laemmli system of SDS-PAGE was
first introduced in 1970s.

4. PrinciplePrinciple
Separates protein in an electric field
Migrate through a liquid or semisolid
medium when subjected to an electric
field from anode to cathode terminal
Molecules flow at different rates depend
on the molecular size of proteins

5.  Sds-coated large proteins migrate slowly through the gel matrix
and small proteins migrate quickly through the matrix
 The nearer the band to the well, the larger the molecular size of
protein

6. What is SDS?What is SDS?
negatively charged detergent sodium dodecylsulfate
(SDS)
used to denature and linearize the proteins
coated the proteins with negatively charged

7. What is PAGE?What is PAGE?
SDS-PAGE is differentiated into two
systems
 Continuous sds-page
 Discontinuous sds-page
Polyacrylamide is used to form a gel, a
matrix of a pores which allow the
molecules migrate at different rates.

8. Polyacrylamide gelPolyacrylamide gel
The size of pores is determined by the
concentration of acrylamide.
The higher the concentration, the smaller
the size of pores.
Discontinuos Sds-Page consists of two
different gels.
Stacking gel (top gel)-4% of acrylamide
Separating gel (bottom gel)- range from 5
to 15%of acrylamide

9. Why polyacrylamide used for a gel?Why polyacrylamide used for a gel?
Chemically inert
Electrically neutral
Hydrophillic
Transparent for optical detection

10. Preparation of GELPreparation of GEL
1.Clean the plates and combs.
2.Set-up the plates on the rack.
3.Pour the separating gel.
4.Pour the stacking gel.
5.Gel storage.

11. Process of SDS-PAGEProcess of SDS-PAGE
1.Boil the samples for 10 minutes to
completely denatures the proteins.
2.Assemble the gel into the apparatus.
3.Pour the buffer solution into the chamber.
4.Load 20uL of samples into the well.
5.After that, run electrophoresis by
connecting the current supplies.

12. Visualization of protein bandsVisualization of protein bands
Visualizes the band under UV light
Types of Stains:
1.Coomassie Blue
Traditional method requires staining followed by
destaining to remove background gel staining.
Most common and least sensitive
Limited to ~100ng of protein
2.Silver Stain
oMost sensitive test
oDetection limit: 0.1-1.0ng of protein

13. A. Staining band with Western blot; B. Coomassie blue stain; C. Silver
stain

14. ApplicationsApplications
Determine purity of protein samples
Determine molecular weight of protein
Identifying disulfide bonds between
protein
Quantifying proteins
Blotting applications

15. Advantages & disadvantagesAdvantages & disadvantages
Advantages Disadvantages
Migration is proportional to the
molecular weight
Poor band resolution due to
high alkaline operating pH
Highly sensitive test, separates
2% difference in mass
Acrylamide gel is potent
neurotoxin chemical
Require small amount of
samples
Gel preparation is difficult and
require longer time
Stable chemically cross-linked
gel
Highly cost

16. ConclusionConclusion
Sds-page is a technique that used to
separate proteins according to their
molecular size through the gel.
Proteins are unfolded and migrate from
cathode to anode terminal at different
rates.
Molecular weight is determined by
compare the result with a standard curve
of relative motility of standard proteins

17. ReferencesReferences
Introduction to Agarose and
Polyacrylamide Gel Electrophoresis
Matrices, written by Patricia Barril and
Silvia Nates
Discontinuous polyacrylamide gel
eletrophoresis of DNA fragments.
Methods in molecular biology. Written by
Budowle, B. Allen, 1991.