PVDF--negative image??

Roney Graf roney.graf at uni-konstanz.de
Fri Aug 21 01:47:33 EST 1998


In article <6ricr0$ho9$1 at nnrp1.dejanews.com>,
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??

   I've seen this occasionally when I had large amounts of the detected
protein in the sample. I figured that by the time I placed the film on the
membrane, all that HRP sticking to it had eaten away the ECL substrate in
the area. The result was a clear spot at the core of the band, with a black
halo around it. Workarounds would be to use less sample, less 2nd antibody,
or to expose immediately after 'immobilizing' the ECL solution on the
membrane (by placing it between plastic sheets, e.g.). 

HTH, 

roney



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