![]() If a single gel is being used for more than one antibody blot, it is convenient to leave a blank well between sets so there is plenty of room to cut the blot later without cutting through samples.Use the Western Blot Template on page 6 to keep track of sample placement and blot conditions. Using gel loading pipet tips, load the required amount of molecular weight markers( 7ul) and samples in designated wells.Loading Molecular Weight Markers and Samples Fill the inner chamber with SDS-PAGE Running Buffer and ensure the chamber doesn’t leak, then fill the outer chamber with SDS-PAGE Running Buffer.If only one gel is being run, place an empty gel clamp into the other side of the electrode assembly. Once the stacking gel has polymerized, insert the electrode assemblie(s) + gel(s) into the electrophoresis tank.Kodak Film X-OMAT Blue XB (PerkinElmer #NEF596).ELC Substrate – warmed to room temperature (GE Healthcare, cat# RNP2132).Horse Radish Peroxidase conjugated Secondary Antibodies.BioRad MiniGel Glass plates, electrode assemblies, blotting sandwiches, blotting sponges and electrophoresis tanks.Transfer Buffer (0.025M Tris, 0.192M Glycine, 20% Methanol).SDS-PAGE Running Buffer (0.025M Tris, 0.192M Glycine, 0.1% SDS).5% BSA antibody solution (5% BSA,0.05% Tween-20 in PBS).5% Blotto Block Buffer (5% Skim milk powder, 0.05% Tween-20 in PBS) **No Azide for HRP conjugated secondary antibodies.Coomassie Blue Stain (0.15% Coomassie Blue, 50% Methanol, 10% Acetic Acid).5X Reducing Sample Buffer (0.25M DTT, 5% SDS, 0.2M Tris pH6.8, 1.5% Bromophenol Blue, 37.5% Glycerol).The blots can be scanned and band densities determined for semi-quantitative analysis. Bands are visualized by adding an enzyme substrate which forms an insoluble, colored product corresponding to the location of the protein band. The membrane is then probed with primary antibodies specific to the proteins of interest followed by enzyme conjugated secondary antibodies. The proteins in the lysates are first separated by size using reducing SDS-PAGE and then transferred to nitrocellulose membrane. Western blotting is a technique used to probe for specific proteins in tissue lysates.
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