[ SDS-PAGE } INTRODUCTION Electrophoresis is the process of migration of charged molecules present in the solution in response to an electric field. Basically electrophoretic techniques have been developed to separate macromolecules on the basis of their molecular weight. The rate of migration of a molecule in an electric field is inversely proportional to the molecular size, shape, viscosity and temperature of the medium and directly proportional to the voltage and charge of the molecule. Proteins could be resolved electrophoretically in a semi-solid matrix strictly on the basis of molecular weight. Under these conditions, the mobility of the molecules would be inversely proportional to their molecular weight. SDS-PAGE technique is exceedingly useful in analyzing and resolving complex protein mixtures. They are employed © Tomonitor enzyme purification ® Todetermine the subunit composition of oligomeric proteins. ® To characterize the protein components of subcellular organelles and membranes. © To assign specific proteins to specific genes by comparing protein extracts of wild type organisms and suppressible mutants.PRINCIPLE Sodium Dodecyl sulphate (SDS or sodium lauryl sulphate) is an anionic detergent which denatures protein molecules without breaking peptide bonds. In PAGE, proteins are uniformly negatively charged by anionic detergent SDS. SDS molecules bind to Proteins and wrap around them by strong hydrophobic interactions and ithas been estimated that 1.4g of SDS binds per gram of protein Polyacrylamide gel is formed by the polymerization of the monomer molecule acrylamide cross linked with N-N'-Methylene Bis- acrylamide (Abbreviated as Bis). Ammonium per sulphate (APS) is used to initiate the polymerization reaction by generating free radicals and a catalyst acting as a oxygen scavenger TEMED (N-N- N'-N'- Tetramethylenediamine) are required to start the polymerization reaction. Denaturation of proteins is performed by heating them in a buffer containing a soluble sulphydry! reducing agent (2-Mercaptoethanol or dithiotreitol) and SDS. Mercaptoethanol reduces all the disulphide bonds of cysteine residues to free sulphydryl groups and heating with SDS that disrupts all intra and intermolecular protein interactions. This treatment to yield individual polypeptide chain which carries an excess negative charge induced by the binding of the detergent SDS. The denatured proteins can be resolved electrophoretically on the basis if size in a buffered Polyacrylamide gel.PROCEDURE 1. Wash glass plates, comb, and spacers. Make sure glass plate are clean, grease free and dry. Assemble the plates as given in the Electrophoresis apparatus instruction manual To a clean glass beaker, add the following reagents to prepare separating gel. Distilled Water - 40m 30% Monomer - 3.3m Separating buffer (pH 8.8) - 25m 10% SDS - 100 pl 10% APS (Ammonium persulfate) - 100 pl TEMED - 5 ul10. 11. 12. Pour separating gel via the gap between the glass plates to % of its length. Ensure no air bubble get trapped in between. Add sufficient amount of 1:1 ratio of toluene on top of the gel to prevent shrinkage of the gel and also avoid dust. Allow the gel to polymerize for 30 minutes. After polymerization of the gel, drain the toluene and dry the remaining toluene by using filter paper. Now prepare the stacking gel as follows: Distilled water - 2.7ml 30% Monomer - 670 ul Stacking buffer (pH 6.8) - 500 ul 10% SDS - 40 ul 10% APS (Ammonium persulfate) - 40 ul TEMED 7 Sul Overlay the separating gel with the stacking gel up to the rim of the notched plate and immediately insert a clean Teflon-comb into the stacking gel solution. Ensure no air bubble get trapped in between. Allow the stacking gel to polymerize for 20 minutes. After polymerization remove the comb carefully. Wash the wells immediately with water. Remove the water by inverting the gel or by using filter paper. Remove the spacer from the bottom and fix the glass plate inthe electrophoresis apparatus filled with running gel buffer in lower and upper buffer tanks. Sample Preparation 13 14 15. 16. 17 18. 19 20. 21, 22. Take three microfuge tubes and mark it as sample |, sample I! and sample Ill Add 10pl of sample |, |! & Ill to the respective tubes and add 10ul of sample solubilizing buffer to each tube. Mix well and boilit at 100°C for 2 minutes, by using water bath. Load the sample |, II & Ill to first three lanes and add 10ul of protein molecular weight marker to the next nearest lane After loading, switch on the power pack and set the voltage at 50V. Run until dye front reaches the separating gel. Then increases the voltage to 100V. Run until the dye front reaches the bottom of the gel. Remove the gel plates from the tank using spatula, carefully separate the gel plates. Remove the stacking gel completely from the separating gel. For identification make a mark on one side of the separating gel. Then place the gel in Stainer and make sure that the gel is completely immersed. Then keep the box in the rocker for 30 minutes at room temperature. And collect the Stainer and store it for reuse. Add 25ml of destainer and allow it for 1 hour with three changes. Observe the clear protein bands as given in the figure.Flow Chart Seal the glass plate with the spacer ‘ Prepare separating gel Pour separating gel to the 3/4" of the glass plate 4 (Allow to polymerize) Prepare and pour the stacking gel upto the rim of the notched plate Immediately place the combs t (Allow the gel to polymerize) Carefully remove the combs and bottom spacer after polymerization ‘ Fix the glass plate in the electrophoretic apparatus Prepare the sample, Add equal volume (10! ) of sample in 10pl of sample solubilizing buffer. 4 (Boil at 100°c for 2 minutes)t Load the samples t Electrophoresis till the dye reaches the bottom ¢ Remove the gel from glass plate Stain the gel by placing in staining solution for 30 minutes Destain the gel by placing in destaining solution for overnight ‘ Observe the protein bands on the gelRESULT AND INTERPRETATION After staining with the Coomassive brilliant blue, the SDS-PAGE gel shows the protein profile of sample |, sample Il and sample Ill The molecular weight of the each sample is compared with protein molecular weight marker 1,16 KDa 66 KDa 45 «Da 29 KDa 14.4 KDa 6.5 KDa Lane #1 Sample! Lane #2: Sample! Lane #3. Samplelll Lane # 4. Protein marker