PVDF--negative image??

Conrad R Fjetland fjetland at bcm.tmc.edu
Thu Aug 27 16:46:41 EST 1998


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

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Hello,

	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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