PVDF--negative image??

[Dr. Peter Gegenheimer]

On Thu, 20 Aug 1998 23:52:00, 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??

We've seen this when the concentration of either primary or seconday antibody
were too high. What I saw looked exactly like photographic solarization; i.e.,
the light intensity was so high that the film image was reversed. In our case
we saw this only in the center of the band, where the light emission would be
strongest. Greater dilution of antibodies, or using a less-sensitive film (a
hand-help Polaroid CRT camera) made the problem go away. So we're pretty
certain the phenomenon was solarization reversal of the film image.

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