Electrophoresis problem

Melanie.Rutherford at dpif.tas.gov.au Melanie.Rutherford at dpif.tas.gov.au
Thu Feb 25 20:24:33 EST 1999


I am not sure if this is relevant to your problem but I was able to greatly
improve transfer of high molecular weight proteins in a semi-dry situation by
adding 0.05% (v/v) SDS to my transfer buffer.  This helps stop the
aggregation of the high molecular weight proteins due to the methanol in the
buffer.  As to the smearing of the protein in the membrane it could be due to
the membrane not being uniformly wetted, the presence of air bubbles, an
excess of heat during transfer or a too rapid transfer of protein (I am
quoting from a trouble shooting page in one of Millipore's "Protein Blotting
Applications Guide" - a good thing for someone like me who is new to this). 
Hope you have some luck.  In article
<g.s.stewart-2402991817270001 at med179.bham.ac.uk>,  g.s.stewart at bham.ac.uk
(Grant Stewart) wrote:

> 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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