1. M.SRI DEVI 2nd Yr, M.Tech BIOTECH
SDS PAGE
M.SRI DEVI
2nd Yr, M.Tech BIOTECH

2. SDS PAGE
Sodium dodecyl sulphate polyacrylamide gel electrophoresis is a type of denaturing electrophoresis, used to separate proteins.
SDS- Sodium dodecyl sulphate
Anionic detergent
SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain coated with negatively charged SDS molecule.
Amount of SDS bound to protein is always proportional to molecular weight of polypeptide.
The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein.

3. PAGE POLYACRYLAMIDE GEL ELECTROPHORESIS
Electrophoresis- study of movement of charged molecules in electric field.
Support medium- polyacrylamide.
Polyacrylamide- synthetic polymer of acrylamide monomers.
SDS- polypeptide complexes migrate through the polyacrylamide gel in accordance with the size of polypeptide.

4. PRINCIPLE
Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the charged R-groups in the protein. The large H's represent hydrophobic domains where non polar R-groups have collected in an attempt to get away from the polar water that surrounds the protein.
After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelms any positive charges the protein had due to positively charged R-groups.
The resulting protein has been denatured by SDS (reduced to its primary structure-amino acid sequence) and as a result has been linearized.

5. PROCEDURE Preparation of plates.
Spacers are kept and sealed with agarose
Separating gel.
Stacking gel.
Sample preparation.
Protein samples made to run in electric field.
Addition of staining solution to gel.
De staining solution.

7. IN DETAIL…….. Separating gel
Acrylamide – 1. When dissolved in water, slow, spontaneous autopolymerization of acrylamide takes place joining molecules together by head on tail fashion to form long single-chain polymers.
2. The presence of a free radical-generating system greatly accelerates polymerization.
3. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another.

8. Bisacrylamide Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels.
2. Bisacrylamide cross links cause the chains to form a mesh like structure in which the holes of mesh represent the pores that retard protein migration through the gel.
Tris HCL – 1. Used to maintain the multiple pH during the process.
SDS – 1. The intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS.
2. Thus polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit length.
3. The electrophoretic mobilities of these proteins will be a linear function of the logarithms of their molecular weights.

9. 4. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass-charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure.
5. This is known as Native PAGE.
6.Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.
Ammonium per sulphate- 1. Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals.
2. An alternative source of free radicals is riboflavin, which generated free radicals in a photochemical reaction.

10. TEMED (N, N, N', N'-tetramethylethylenediamine)- 1
TEMED (N, N, N', N'-tetramethylethylenediamine)- 1.TEMED a free radical stabilizer initiates polymerization of ammonium per sulphate.
2. The rate of polymerization and the properties of the resulting gel depend on the concentrations of free radicals.
3. APS and TEMED are typically used at approximately equimolar concentrations .

11. DIFFERENCE????? Stacking gel Acrylamide Bisacrylamide Tris HCl SDS APS
TEMED
1. Pore size of
the gels.
2. pH.
DIFFERENCE?????

12. 4. Due to low pH they lose their charge and slow down.
Running buffer
Glycine Weak acid and exists in either of 2 states - uncharged or charged glycinate anion.
5. Chloride ions also move away from the cathode, thus leaving a high electric field.
2. At low pH it is protonated and thus uncharged at higher pH it is negatively charged.
3. When power goes on the glycine moves away from the cathode and head towards sample and stacking gel.

13. 6. This helps the highly negative charged protein to move towards the anode. 7. The effect of moving in high voltage is all proteins reach the separating gel at the same time, so migration is truly a function of molecular size. 8. When they reach the separating gel everything changes. 9. The pH goes up and glycine becomes deprotonated making them to move fast. 10. The protein thus moves in a slow and relaxed manner.

14. Gel loading buffer
Glycerol – 1. It is used to obtain higher viscosity of the gel, to make the handling with it easier.
2. Makes the sample more dense than the sample buffer, so the sample will remain in the bottom of a well rather than float out.
Tris HCl - 1. Cl used to
facilitate glycine.
SDS - 1. Used for
denaturation of sample.
2. DTT used as alternative.

15. β- Mecaptoethanol - 1. A reducing agent with the formula HSCH2CH2OH
2. Acts to break disulfide bonds between cysteine molecules.
Bromophenolblue(3',3",5',5"tetrabromo
phenolsulfonphthalein)
1. Used as indicator.
2.Being highly mobile molecule it moves
ahead of all proteins .
3. Weakly bind to some protein and impart
Blue colour.

16. Staining solution CBB R250(Coomassie brilliant blue) C45H44N3NaO7S2 –
methanolic solution acidified with acetic acid.
CBB R250(Coomassie brilliant blue) C45H44N3NaO7S2 –
Acetic acid - 1. Proteins in the gel are
fixed by acetic acid
1. Protein stain.
2. Anionic dye, which non-specifically bind to protein.
and simultaneously stained.
Methanol - 1. Fix protein in gel.
3. The structure of CBB is predominantly non-polar, and it is usually used in

17. Therefore, large volume of staining solution is recommended, at least ten times the volume of the gel.
Destaining solution
The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye.
The proteins are detected as blue bands on a clear background.
As SDS is also anionic, it may interfere with staining process.

18. RESULTS…..

19. QUERIES????