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        SDS-PAGE

        互联网

        1815

        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)

        Percentage of gel 8% 10% 12.5%
        30: 0.8% w/v acrylamide:bisacrylamide 2ml 2.5ml 3.1ml
        1.0M Tris-Cl pH 8.8 3ml 3ml 3ml
        20% SDS 38μl 38μl 38μl
        dH2O 2.43ml 1.9ml 1.3ml
        Mix together.Add APS and TEMED just before pouring      

        10% APS 36μl 36μl 36μl
        TEMED 5μl 5μl 5μl
          7.5ml 7.5ml 7.5ml

        STACKING GEL (NOTE pH 6.8)--use 4% stack for <10% res.gel and 6% stack (easier to handle)for >10% resolving gels.

        Percentage of stack 4% 6%  
        30:0.8% w/v acryl:bisacryl 660μl 1ml  
        1M Tris-Cl pH6.8 630μl 630μl  
        20% SDS 25μl 25μl  
        dH2O 3.6 ml 3.6 ml  
        Mix together.Add APSand TEMED just before pouring.      

        10% APS 25μl 25μl  
        TEMED 5μl 5μl  
          5ml 5ml  

        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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