Per.Bergman at vf.slu.se
Fri Jan 12 05:26:43 EST 1996
I have some problems with my western experiments. I am analysing
rapeseed and tobacco
protein extracts by the westerns-blot technique. I have an interest
in mitochondrial proteins and genes
affecting flower development. As you might know plant mt-genomes
are large (200-400 kb) and contain many genes not found in mammals. The
anti-sera are produced in rabbits and the injected
antigen is either GST/protein fusion's of synthetic peptides based
on genes whose expression we can correlate to flower mutant phenotypes.
My problem is that I consistently get 10 or more, quite strong,
background bands. We use the ECL-system for detection. This problem
prevails even if I use a "known antiserum". With my floral mutants I only
get these background bands, even with "pre-immune sera".
Is it possible that the carbohydrate molecule bound to my IgG is
present in the protein preparations????
I get background if I use pre-immune sera + secondary
goat-anti-rabbit ab also.
Has anyone seen this before, and HOW did you solve the problem. I
will try to soak the
filters with normal goat serum, to block any lectin binding, if
this is the case.
Post suggestions here or mail me at: Per.Bergman at vf.slu.se
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