Electrophoresis problem

Lena Zaitseva zaitseva at biochem.purdue.edu
Fri Feb 26 09:38:45 EST 1999


     In our lab we are running discontinuous gel electrophoresis using
following solutions:
1. 4 x Tris-Cl/SDS, pH 8.8 (1.5 M Tris-Cl containing 0.4% SDS)
2. 4 x Tris-Cl/SDS, pH 6.8 (0.5 M Tris-Cl containing 0.4% SDS)
3. 30% acrylamide/0.8% bisacrylamide.
     The first buffer (pH 8.8) is used for the separating gel (bottom part).
Another buffer (pH 6.8) - for stacking gel (upper part). It is described in
details in Current Protocol in Molecular Biology.
     For transferring proteins to the membrane some SDS is included in transfer
buffer:
48 mM Tris
39 mM Glycine
0.037% SDS
20% Methanol,
adjust pH to 8.4 with dilute HCl (usually it is not necessary)
    It is probably worth to increase SDS concentration (up to 0.05-0.1%) to
improve transfer of high molecular weight proteins.
 
     Lena.
 

Grant Stewart wrote:

> 6% acrylamide gel:
>
> 27.4mls H2O
> 4mls 1M Tris/1M Bicine Solution
> 8mls 30% Acylamide solution (37.5:1 Acrylamide:Bisacrylamide)
> 400ul 10% SDS
> 80ul TEMED
> 200ul Fresh 10%APS
>
> Wet Electrotransfer Buffer:
>
> 7litres
>
> 203g Glycine
> 40.6g Tris
> 20% Methanol.
>
> Yours sincerely,
>
> G.Stewart

 
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