SDS-PAGE: gel electrophoresis of proteins TECHNIQUE is to set up gel plates before you mix the gel mixes.Use the THIN spacers and choose a comb--number of wells varies.Make both resolving gel and stack (NO APS or TEMED)--then add APS and TEMED to resolving gel,mix and pour about 3-3.5ml per gel.Before it polymerises,now add APS and TEMED to your stack mix and pour it gently on top of the resolv.gel (using a pasteur pipette works well).Put in the comb and let solidify.Should take 15-20 minutes.You can keep gels overnight at 4℃ if well wrapped to prevent drying out and if you keep the comb in.Note: just before you want to load gels,wash out wells with distilled water to remove unpolymerised acrylamide. GEL RECEPIES (enough for 2 thin mini-gels) RESOLVING GEL (NOTE pH 8.8)
STACKING GEL (NOTE pH 6.8)--use 4% stack for <10% res.gel and 6% stack (easier to handle)for >10% resolving gels.
Preparations of sample: Protein samples are in 1X sample loading buffer (also called Laemmli dye)...ie to 6μl protein sample,add 3μl 3X Laemmli dye stock.Boil 3 minutes before loading gel. Sample loading buffer (Laemmli loading dye)3X stock: 1M Tris-Cl pH 6.8 2.4 ml 20% SDS 3 ml Glycerol (100%)3 ml B-mercaptoethanol 1.6 ml Bromophenol blue 0.006g 10 ml (store 4℃) 10X Running buffer (also called Laemmli buffer): Tris base 30.3 g Glycine 144 g SDS 10 g make to 1L with dH2O For BioRad apparatus: need 500 ml of 1X buffer so dilute 50 ml 10X stock + 450 ml dH2O. Running gels: Using the BioRad apparatus,run gels at 200V (constant voltage)until the bromophenol blue dye is just off.Takes 50-60 minutes.Gels are run at room temperature. |
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