tearing my hair out over IPs
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.
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