Re-ECLing nitrocellose??

T. Max M. Soegaard tmms at mbio.aau.dk
Thu Nov 2 16:46:22 EST 2000


Thats strange, In my lab we currently are experiencing problems with the
ECL.
It gives rise to "clouds" on the film so what people do when they see
this is to  simply rinse the blotted PVDF-membrane in dd-water or PBS
and re-apply ECL. After that the blot is fine. No clouds. But mind you,
this is on wet - non-frozen blots.
-Max

Paul wrote:
> 
> Neal Robert Melvin wrote:
> 
> > Has anyone ever tried to 're-do' an ECL reaction on an NC membrane that
> > has been previously processed, and subsequently dried?? Do you have to
> > strip the membrane first, and re-apply primary and secondary antibodies
> > even if you want to re-probe with the same primary as you used
> > originally??
> >
> 
> We do this fairly often: we don't bother stripping but we've found that we
> 
> do have to re-apply the primary and secondary Abs.  Dumping fresh ECL
> substrate on without fresh Abs doesn't work in our hands, even on a fresh
> blot that's not dried out.  Not sure why, but the guess is that the HRP
> becomes inactivated.
> 
> Cheers
> 
> Paul Digard






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