background problems with Abs

GRANT at CUCCFA.CCC.COLUMBIA.EDU GRANT at CUCCFA.CCC.COLUMBIA.EDU
Fri Apr 21 15:53:00 EST 1995


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 
contaminating Abs?  

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:

Sincerely,

Barth Grant



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