dephos with alkaline phosphatase - (Jan/31/2008 )
my goal is to test an AB which is supposed to recognize a phosphorylated form of my protein.
So the plan is to lyse my cells in RIPA, and then treat half of the lysate with alkaline phosphatase (AP, ROCHE), and not the other half. The question is: would this work in RIPA buffer? do I just need to add the AP or do I need to add something to my RIPA lysate? Does anybody has experience with that kind of experiment?
The second part of the experiment will be to IP with dome AB and then +/- treat with AP, and WB with my anti phospho prot AB.
same question: which buffer should I use, compatible with regular sds page gel.
If you have any info or suggestion, I would greatly appreciate (I am not a protein person at all ).
our lab has tried to dephosphorylate a substrate of an enzyme. But the buffer conditions were different. We had tris, Nacl in the buffer and no detergent. Even having some triton might not affect AP but RIPA, i am not sure. Even in SDS, AP will not work. This was in our hands.
I got the same answer from another source: no way to dephos in total lysis buffer with AP. or do it without anti phosphatase inhibitors and work in a dirty way... not too scientific
Will go for IP, too bad!