University:
Swansea University
Course:
PM134 | Microbiology
Academic year:

2023
Views:

312

Pages:

2
Author:

Sanjeev K

XPERIMENT No:4 DATE: 20-10-2022 SDS-PAGE POLYACRYLAMIDE GIEL ELECTROPHORESI AIM Separate proteins, by SDS PAGIE PRINCIPLE Palut separates, protein modeutes on the basis of both size and change, through change, in an electric fiels They migrated a liquilid Cainede terminal. Mokrile flour nding 01 Lerrusoled medium on the mobulantemules of proteins Proteinm from to diff brent nates, depe both aide and banc groups? be kept are amphoteric compounds, containing The PH constant to maintain the the solution Enarge and to 9 protein For this reason the proteins are Electrophorused it Buffers SDS-wated large protein large protein mobility gel matrix and shau protein migrate gilickly throu the matrix. me here to band to the well, the larger the molecular inzy protein MATERIALS REQUIRED Electrophoretic tank, spous, Powerpack, clips, glass plates, micropipette, acrylamide, glyeine tus, bromophend blue, butand, 4, bis acrylamide, AP'S, TEMED, SDC bue, glycad, coomarsie brilliant B-morcapro etnarld, solution, ged illuminator, agar PROCEDURE 1, staining af destaining * Wash all the avessones of gel apparatus with soap, sive thoroughly, allow to air dry * place the 2 plates on the bench kap and left edge kap eve spacer al righe two spaws al the rig the bottom * Seal the plate with 1% agar en the tinee sides. * Place the notch plate of the top and align I align of the plates * Fre the aligned glass plates to the Clamps assembly Pour distilled water to the awembly mun flantakage Felter paper is used to absorb water between the glass plate * separating gel is prepared and powsd between the plates up to 3/4 height and left for 30 minutes Meanwhile, Marking gel is prepared and it is poured be plates above ween the glass plates! ely comb is placed. the separating gel. Immedi 8 * Allows the gel to Then the Glass polymorize for. 30 minutes tank, that contains ed inside the electrophoretic are placed * comb is removed cabulier and the given samples are, and connected to powerpark. loaded with in well. * Put the with micropipette. In each in the power supply which is sun initially at sov followed by! * Once the bromophenol bule the reaches the bottom of 100%. the gel, dissonneet the power supply * For staining, immerse the gre the tray on a in the staining solution of place slowry rotaring platform/snedker for A howrs at room temperature. a min.f * Destain the gel by plaung it in the destaining solution. Pla the tray on how shaker for another 4-8 hours. Change the destaining solution 3-4 times * Take a picture g illuminator. the stained gel after plaung it on gel RESULTS The given sample migrated towards the oppositely Charged side under the inference of different rates, this was electric field al compared with the marker