help ponceau staining
Thu Dec 23 13:48:41 EST 1993
In article <1993Dec22.230418.1 at vms.ucc.okstate.edu>,
zeljkos at vms.ucc.okstate.edu wrote:
> I am having troubles using Ponceau S solution for temporary staining of
> western blots.
> I am definitely sure that my proteins were transferred to the membrane
> (rainbow marker confirms that) and in the satisfactory quantity.
> The Ponceau S (Sigma) solution was always prepared fresh according to
> Current Protocols in Mol. Biol., what means: 0.5g of Ponceau S dissolved in
> 1ml of acetic acid and diluted to 100ml with ddH20.
> The membrane used were PVDF either from Sigma or Biorad.
> I have tried with various incubation times from 5 to 30 minutes. Destining
> was under stream of the ddH20.
> Directly after semi dry transfer, the membrane was rinsed with PBS
> (50mM KP, 100mM NaCl) and immersed into Ponceau S solution.
> PLEASE DO SOMEONE HAS IDEA WHAT WAS WRONG. Thanks Zeljko.
I think your problem is that you destained too much. Ponceau S is a
reversible stain that is removed with water. What you can do is just
briefly immerse (DO NOT PUT UNDER STREAM OF WATER!) in water with gentle
shaking and watch carefully. The destaining only takes a minute or so.
Boyce Thompson Institute
E-mail: pw15 at cornell.edu
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