SDS PAGE

M.SRI DEVI2nd Yr, M.Tech BIOTECH

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.

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.

PRINCIPLEProtein (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.

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.

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.

Bisacrylamide - 1. 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.

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.

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 .

Stacking gel

Acrylamide

Bisacrylamide

Tris HCl

SDS

APS

TEMED

1. Pore size of

the gels.

2. pH.

DIFFERENCE???

??

Running buffer

Glycine - 1. Weak acid and

exists in either of 2 states -

uncharged or charged

glycinate anion.

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.

4. Due to low pH they lose

their charge and slow down.

5. Chloride ions also move

away from the cathode, thus

leaving a high electric field.

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.

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.

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.

Staining solutionCBB R250(Coomassie brilliant blue) C45H44N3NaO7S2 –

1. Protein stain.2. Anionic dye, which non-specifically bind to protein.3. The structure of CBB is predominantly non-polar, and it is usually used in

methanolic solution acidified with acetic acid.

Acetic acid - 1. Proteins in the gel are

fixed by acetic acid and simultaneously

stained.

Methanol - 1. Fix protein in 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.

Therefore, large volume of

staining solution is

recommended, at least ten

times the volume of the gel.

RESULTS…..

QUERIES????