Background problems. or Why are my worms so dirty?
I would like to find out what methods have been
successfully implemented to "clean up" polyclonal
antibodies for use in whole mount staining of worms.
I have affinity purified a rabbit polyclonal
generated against fusion protein. On western blots
of worm extract I can detect a strong band of the
expected size (which is not present in a null
mutant). This purified Ab doesn't give much
background on westerns at all, but on "Finney &
Ruvkun" fixed worms the background (as determined by
comparing WT with the previously mentioned null) is a
real problem. I have tried incubating the affinity
purified Ab in an E. coli acetone powder as a
negative selection. This had little to no effect.
Has anyone tried to make a C. elegans acetone powder?
Has preincubating antibodies with fixed worms removed
P.S. I know that the secondary is fairly clean. But
if anyone can suggest a source of secondarys that
they think is really good, I could probably benefit
from your experience on this front also. I would
also be curious to know which conjugate gives the
highest signal:noise ratio.
Any suggestions would be greatly appreciated: