tearing my hair out over IPs

AShaw5 ashaw5 at aol.com
Fri Apr 12 00:01:40 EST 1996


I have a few suggestions.  
I would lyse in something more stringent.  We generally use something with
1% NP40 and 0.6% deoxycholate. Leave the cells on ice for 5 to 10 minutes.
 It has been reported that cytoskeletal inhibitors can help to reduce the
background at this step.  After spinning out the nuclei, you can then add
SDS to 0.3%.  You can try reducing the time of incubation as well as
increasing the temperature (37 degrees).  Try washing more stringently. 
Some groups pellet the proteinA complex through a sucrose cushion. 
Finally, it is possible that your control serum is bad or very sticky. 
The better control would be to use the protein immunogen (hopefully a
peptide) to block the binding and complex formation.  With your
overexpression studies, if you can show that antibody to protein x does
not crossreact with protein y you should now be in business.  Good luck.

Andrey Shaw



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