western blot: colorless protein bands

Bernard Murray, PhD spam at 127.0.0.1
Tue Feb 29 23:22:23 EST 2000


In article <01JMHKHPYAJ60002N2 at if1.if.ufrgs.br>, ita at dna.cbiot.ufrgs.br
(ITABAJARA DA SILVA VAZ JUNIOR) wrote:

> In a Western blot the nitrocellulose was blocked with 5% blotto 
> and an anti-mouse IgG-phosphatase alkaline conjugate was 
> used.
> These produce a negative image of colorless protein bands 
> against a clear background.

I've seen "negative bands" in situations where the signal
was waaaaaay too strong (usually when I was testing the best
titre for the antibody and the best amount of antigen to load).
In the absence of any other information about your blotting
conditions this would be my first guess but I could be quite
wrong.  If this is the case then drop the concentration of
primary antibody and/or the amount of protein you are loading.
It is unlikely to be a problem with the secondary antibody
as your background sounds okay.
   Good luck,
      Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF




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