sci-mmw at jcu.edu.au
Thu Aug 27 20:53:50 EST 1998
I have also experienced the problem you refer to of a negative image when
using ECL. When you have heaps of target molecule, which in turns binds
heaps of your tagged detecting molecule, the ECL signal is quenched before
an image appears on your film. Try blotting a dilution series of your
proteins onto the PVDF. The resulting probe should identify which is the
optimal protein/detection system concentration when using ECL.
On 27 Aug 1998, Conrad R Fjetland wrote:
> stebby at welchlink.welch.jhu.edu wrote:
> : Help...please!
> : A friend just called to ask me to troubleshoot a commercially purchased
> : western blot on PVDF which, when stained for ECL, yielded clear zones where
> : the proteins in each lane are located on the blot. No signal was detected,
> : rather they had what amounted to a negative image of what a coomassie stained
> : gel would have looked like. It sounds like the protein bands were somehow not
> : accessible to the antibodies. I don't use PVDF so I'm stumped already! Any
> : takers??
> : FYI--blots made in their lab onto PVDF (I don't know which supplier) were
> : used to determine the optimal primary and secondary antibody dilutions prior
> : to their use of the commercially obtained blot. The know the antibodies work
> : and the antigen should be there.
> : Thanks to all!
> : Steve Dahl
> : JHMI
> : -----== Posted via Deja News, The Leader in Internet Discussion ==-----
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> Do you see brown spots on the PVDF membrane that corresponds to
> the spot on the film. If you do, then the reaction is already over. Try
> a lower dilution of the primary and secondary ABs. If this is not the
> case and you just are not seeing anything where there is a band on the
> Stained protein gel, it could be another protein masking yours. This is
> overcome by altering the concentration of the gel of changing the pH of
> the sep buffer. Hope this helps.
> Conrad R, Fjetland
> Baylor College of Medicine
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