BT 441

Expt No: 1

Aim: To separate and determine the molecular weight of the proteins by using Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Principle: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), is a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility,a function of length of polypeptide chain or molecular weight. In SDSPAGE, proteins are separated according to their charge to mass ratio by sieving action of polyacrlylamide gels. When proteins are treated with SDS and Beta-mercaptoethonol or DTT they will have uniform charge to mass ratio with random coil form, because the amount of SDS that binds to the protein is proportional to the mass of the mass of the protein i.e., 1.4gm of SDS will binds to 1gm of protein or one molecule and SDS binds with two residues in proteins and results in fractionation by size. Reagents: SDS-PAGE S.No Reagent 4%Stacking gel 1. 30%acrylamide/bis-acrylamide 0.264ml (30:1)or(29:1) 2. 0.5M tris-HCl(pH 6.8) 0.504ml 3. 0.5M tris-HCl(pH 8.8) 4. 10% SDS 20ul 5. Distilled water 1.2ml 6. TEMED 2ul 7. 10%APS 10ul Total 2.000ml 12%Resolving gel 1.6ml 1ml 40ul 1.34ml 2ul 20ul 4.002ml

Commassie Staining: S.No %gel 1. 2. 3. 4. 7% 10% 12% 15% Mol.wt range 50-500 K.Da 20-300 K. Da 10-200 K.Da 3-100 K. Da S.No Component 1. 2. 3. Acetic acid CBB Distilled Water Quantity 10%(V/V) 0.006% 90% For 1 Lt 100ml 60mg 900ml

Mix overnight and filter using filter paper

Fixing solution For Coomassie Staining:

S.No Component

Quantity

For 1 Lt Destain : S.No Component Quantity For 1 Lt 45% 10% 45% 100ml 250ml 650ml

1.

Acetic acid

10%(V/V)

100ml

2. Isoproponal

25%

250ml

1. 2.

Methanol Acetiacid Distilled Water

3.

Distilled Water

65%

650ml

3.

The technique consists of three basic steps: 1. Preparation of PAG: Crosslinked PAG is formed by copolymerization of acrylamide monomer and a crosslinking agent N-N-Methylene bisacrylamide. This reaction is catalysed by Tetra Methylene Diamine(TEMED) and intiated by Ammonium Per Sulphate(APS). Porosity of the gel is determined by the amount of acrylamide and bisacrylamide used. Poly Acrylamide Gels are described by two characteristics: (i) Total monomer concentration: (%T=(acry+bis-acry)/total vol. X 100) (ii) Crosslinking monomer concentration: (%T=(bis-acry/(acry+bis-acry) )X 100) In mixing solution 30% (W/V) stock solution corresponds to 30%T. %C remains constant at all dilutions and cannot be readjusted. Where as %T will vary depending on the amount of the water added. 2. Electrophoresis: Polyacrylamide gel slab is prepared and fixed to a vertical electrophoresis unit. Protein samples are denatured by boiling in sample loading buffer. Differences in the pH and composition between stacking and resolving gel causes the sample to be concentrated in to narrow bands by isotachophoresis. When dye reached to the bottom of gel, electrophoresis is stopped. 3. Visualisation: Proteins are colourless and hence cannot be visualized directly. Suitable dye such as commassie brilliant blue R-250, silver staining is generally used to stain the proteins resolved on the gel. Procedure: 1. Clean the gel glass plates with 50% ethanol and arrange the apparatus and check for leak proof with distilled water. 2. Prepare stacking and resolving gels according to the quantities mentioned. 3. Add TEMED as the last step of gel preparation to avoid polymerization prior to stacking.

3. Pour 4ml of resolving gel in the stacked apparatus and allow it for polymerization. 4. Add ethanol or water to avoid atmospheric oxidation and then pour the stacking gel after removing the ethanol or water. Insert the comb between the plates. 5. Allow the stacking gel also to polymerize and then remove the comb. Place the gel stack in the buffer tank.. 6. Mix the test protein sample with equal volume of gel loading buffer in eppendroff and mix it uniformly followed by boil at 1000 C for 10 mins. 7. Load the protein sample into the wells by using micropipette or with HPLC syringe between the plates and Pour the buffer and then initiate the electrophoresis at ~ 75 volts. 8. After the gel run is completed remove the thin plate, cut off the stacking gel and incubate the resolving gel in fixing solution for 15mins. 9. Fix the gel in staining solution (Commassie brilliant blue R-250) and allow the gel for overnight staining. Remove the CBB and destain (Methanol, acetic acid and water 45:10:45) the gel until the protein bands are clearly visible under transilluminater. 10. Measure the distance migrated for each band and thereby calculate the R.F value for each sample. Relative Mobility: Use a ruler to measure the distance migrated by each band of the protein from the start of the separating gel. Relative Mobility of proteins (R.F) = (Distance migrated by protein in cms)/ (distance migrated by the tracking dye in cms)

Result: