a recipe of a lysis buffer for co-IP (phosphorylation) - can anyone recommend one? (Apr/28/2005 )
different people have different lysis buffer for co-IP, so I can't make a good choice. can anyone recommend one good for assaying phosphorylation of p38 MAPK?
Thanks in anticipation.
Qiagen has CHAPS as detergent in their lysis buffer for their Phosphoprotein purification kit. I have tried this lysis buffer with great success, however, I have added Octyl D glucoside 1% which is non-dnaturing detegent with high CMC (critical Miscelle concntration) to ensure solublizing most if not all membrane proteins such as MAPK and they will not interfere with your Co-IP experiment, along with a phosphatase inhibitor such as sodium vanedate. Good Luck!
could you tell the details?
I have used 1% w/v CHAPS and 1% w/v Octyl D glucoside in PBS buffer for the phosphatase sodium vanidate I have used 1mM as ageneral phosphatase inhibitor, however, you can check CalbioChem and Sigma as they offer phosphatase inhibitor cocktails. good Luck!