Electrophoresis problem

Holger Neumann holger.neumann at ruhr-uni-bochum.de
Wed Feb 24 18:02:33 EST 1999


Hi Grant Stewart,

you 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: [snipped]

I personally think you have problems transferring your protein to the
nitrocellulose membrane.
Perhaps this can help:
1. run SDS-Page
2. stain SDS with Coomassie
3. document your stain on photo (digital or conventional)
4. destain SDS-Page
5. Western blot proteins
6. stain membrane with Ponceau
7. document this second stain like the first one (see 3.)
8. evaluate both pictures to see where the problem occured

if bands are smearly in Coomassie stain your SDS run makes some problems
if bands are clear in Coomassie but smearly in Ponceau stain you have problems
transferring the protein

Good luck

Holger
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M.D. student  Holger Neumann
University of Bochum
Germany
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