Electrophoresis problem

Grant Stewart g.s.stewart at bham.ac.uk
Wed Feb 24 13:17:27 EST 1999


Can anyone help...?

I am mailing concerning problems our institute is having running SDS page
acrylamide gels. We have found that we are losing proteins in the gels (as
seen when using prestained markers). When the gels are transfered then
there is a lot of protein left in the gel when Coomasie stained and on the
nitrocellulose the Ponceau S stain show smeary lanes with no defined bands.
It seems to be the higher molecular weight proteins which are most highly
affected. We have changed the Water, SDS solution, Tris/Bicine stock
solutions, Acrylamide/Bisacrylamide solution and powder, APS and Temed.
None of the results show any consistency therefore we have not been able to
narrow down the problem(s). We have also used plastic wear instead of our
own cleaned glasswear as we thought that it maybe some residue retained on
the glasswear from cleaing that maybe affecting the gels. This has not been
reproducibly shown to make any difference. We also have experienced
variablility in wet and dry transfer but we think that this maybe down to
problems with the gel and not with the transfer itself although this maybe
wrong. If you could provide any suggestions then we would be very grateful
as we don't know what else it could be. The recipe for the gel mixture and
transfer buffer are given below:

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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