Co-IP in sonicated whole cell lysates - (Dec/05/2007 )
have anybody tried co-ip on sonicated lysates. i have trouble getting sufficient nuclear proteins out in my conventional lysing. can sonication distrupt nuclear protein interactions?
few hints would be helpful
Yes, I have tried. It worked. But you need to add much more protein to
account for the large increase in histones that will be in you sample.
When I IP for chromatin-bound material I either lyse cells with 1% detergent and 420mM NaCl
and then dilute this at least 5 times in IP buffer. The detergent and high salt will
extract many nuclear proteins.
Alternatively you can do an MNase digestion of your nuclei and then collect the nuclear proteins as this is less harsh than sonication.
I have bad experiences with sonication. If not properly set, it can distrupt anything, particularly the interacions you want to keep... If the sample is heated in process, it denatures the proteins, so high volume of your sample is critical.
I'd really prefer digestion.
Thx for the hints....will try it!