SDS PAGE Analysis

EXPERIMENT 7 SDS. :20.10.2021 AIM PAGE POLYACRYLAMIDE GEL ELECTROPHORESI PRINCIPLE Separate Proteins by SDS PAGE PAGE size separates protein molecules on the basis of both through and a charge, in an electric field They migrate anode to cathode liquid or semisoled medium from dept nding on terminal the molaular Molemules size flow proteins at diff event rates. be kept constant to maintain groups the Enarge mobility in both audic Proteins and are basic amphoteric of The compounds, pH of the solution and containing has to buffers SDS-wated large proteins migrate slowly throug of Proteins For this reason, the proteins are electro phoresed through t. the matrix. The nearer the band to the well, the larger get matrix and small proteins migrate quickly the molecular sizig protein MATERIALS REQUIRED Elutrophoretic tank, spaces, powerpack, clips, glass plates, micropipette, acrylamide, bis airylamide, APS, TEMED, DS glycine ,tris, bromophend blue, glycorol, coomassie brilliant blue, butand, B-mircapto enand, staining as destaining solution, gel illuminator, agar PROCEDURE Wash all the accessones of gel apparatus with soap, rinse thoroughly allow to air dry place the 2 plates on the bench keep two spaurs al the right and left edge keep one spaural the bottom Plau the notch plate on the top and align Seal the plate with 1% agar on the three sides of the plates in the aligned glass plates to the clamp assembly Pour distilled water to the assembly and check for leakage Filter paper is used to absorb water between the glass plates separating gel is prepared and poured between the plates up to 3/4 height and left for 30 minutes and It Meanwhile, stacking gel is prepared is poured be ween the glass plates above the separating gel. Immed um comb is placed

8 Then Allow the the gel to polymerize 30 minutes tank, comb that glass contains plates are placed for and connected inside the to electrophoretic powerpark buffer is Put loaded run the is with lid removed on micropipette the carefully tank in up and turn each the on the well. given power samples supply are which disunnet Once the initially bromophenol the at 50V but followed the reaches by 100v. the bottom of the gel, For staining, immerse the supply get in the staining solution a al place power the tray on a slowly rotating platform/shaker for min.g 4 hours at room temperature Destain the gel by plaung it in the destaining solution change Pla the tray on slow shaker for another 4-8 hours. Take the destaining a picture of solution the stained 3-4 times gel after planing it on gel illuminator. RESULTS The given sample migrated towards the oppositely charged side under the influence of electric field at different rates, this was compare with the marker good