SDS-PAGE is a technique used to separate proteins . The concept of stacking and resolving gel in this SDS-PAGE are briefly describe in very interesting and easy way to understand the role of stacking and resolving gel.

1. Presented by -Kumari Jyoti
Msc in biotechnology and diploma
in forensic science
Vinoba bhave university
Topics- SDS-PAGE

2. Sodium dodecyl sulphate -
polyacrylamide gel
electrophoresis.
(SDS-PAGE)

3. Principle
Material and method's
Applications

4. Principle of SDS-PAGE
SDS- Electrophoresis is a technique used to separate protein
molecules according to molecular wright.Protein is negatively
charged(due to SDS detergent) and when electric field is applied
across the gel ,protein migrates through the gel is dependent upon
-
1. Molecular weight of protein
2. Agarose- concentrations
3. Applied current

5. Materials used in SDS
electrophoresis and its
function.

6. • Polyacrylamide gel-
• Reason behind polyacrylamide used is -
• It is inert
• Electrically neutral
• Transparent
• Crosslinked polyacrylamide gel are formed by co -polymerization of acrylamide
monomer and a cross linking by N,N' - Methylene-bis-acrylamide.
• Ammonium per sulphate is used as initiator.
• TEMED is used as catalyst.
• Lower percentage of gels-favour larger pore size -it separate larger molecules
• Higher percentage of gel -favour smaller pore size -it separate smaller molecules.

8. Buffer- SDS -PAGE utilise
discontinuous buffer
system.
Buffer systems include the buffer used to-
• Cast the gel
• Prepare the sample
• Running buffer preparation

9. Buffers used in SDS - PAGE are-
Tris glycine(pH=8.3)
Tris HCl(pH=6.8)-
Tris HCl(pH= 8.8)-
Tris glycine(pH= 8.3) is used in
running buffer.
Tris HCl (pH=6.8) used in stacking gel
Tris HCl (pH= 8.8) is used in
separating gel.

10. SDS-
Sodium dodecyl sulphate is an anionic detergent.
It binds strongly to proteins,causing their
denaturation.
SDS make the protein negatively charged.

11. Beta marcaptoethanol -
Is used to cleaves the disulfide bonds.
Breaks the disulphide bond
present in protein
Protein

12. Steps in SDS-PAGE

13. Steps-
1. Gel preparation.
2. Casting the gel.
3. Loading the samples
4. Observe the protein bands
5. Results interpretation.
Stacking gel
Separating gel

14. Polyacrylamide gel preparation for
stacking gel
Acrylamide- less concentration
0.5M Tris (pH=6.8)
SDS
dH2O
APS(ammonium per sulphate)
TEMED

15. Polyacrylamide gel preparation for
resolving gel/separating gel
Acrylamide-high concentration
1.5 M Tris (pH=8.8)
SDS
dH2O
APS(ammonium per sulphate)
TEMED

16. Firstly we pour the resolving gel in gel caster
After the gel is set we place the comb in caster
After the gel is set we pour the stacking gel above
the resolving gel or separating gel.
Add running buffer to cover the surface of the gel.

17. Sample
preparation

19. Bromophenol blue dye role-
It is used to track the
protein in gel during
electrophoresis

20. Invisible (protein is
colurless ) Treated with
dye
Protein is
visualize

21. Dye is used to visualize the protein just like the Mr
India invisible concept-
When invisible watch is activated it make the wearer
invisible to naked eye but when red light is focused on

22. SDS (anionic detergent) make
the protein negatively charged
Protein

23. Beta marcaptoethanol-role

24. Without glycerol the
sample is less dense so it is
not settled in well
Glycerol provide the
density to the sample

25. Role of
stacking gel

26. Stacking gel play a role to stack protein at
one position.

27. Stacking gel is used to stack the protein in one place so
that they all enter in resolving gel at same time,just
like the marathoners stack in one position before the
run.

28. Role of separating gel

29. Resolving gel also called separation gel is used to
separate protein molecules according to their mass.

30. Figure show that sample enter in separating gel and
1. Chlorine reaches the ist position
2. Glycine in second
3. Protein is third position

31. Staining dye-
Add the coomassie blue to visualize the bands with clear
background.

32. Gel doc system is used to visualize the
Protein bands.

33. Result- protein bands

34. Video link for SDS PAGE
https://youtu.be/eaETFKXtNRA

35. Applications of SDS-PAGE
• It is used to measure the molecular weight of the
molecules.
• It is used in peptide mapping
• It is used to estimate purity of proteins.
• It is used for sample preparation in prior to
spectrometry

36. Thankyou