[Bioforum] immunoprecipitation problems
(by Hongbo_Gu from brown.edu)
Wed Dec 5 09:42:53 EST 2007
I recently met an problem with IP, I tried 2 commercial Abs try to pull down one membrane receptor, but both of them falied, but they can successfully pull down this receptor in the mimic experiment (I have some recombinant receptor proteins expressed from E. Coli), I have tried different lysis buffer, different amount of Ab and other conditions. Because the MW of this receptor is about 25KDa, which is closer to the light chain of Ab, so I used protein G-HRP to detect the receptor by western.
I am wondering is there something wrong with sample preparation, I added the lysis buffer to cells, sonicate them briefly, incubate on ice for 1h, centrifuge and pre-clear it by preimmune IgG and protein G bead, after all above steps, the receptor still present by western, but cannnot be pulled down by IP.
The only reason I could think of is that the potential PTMs of the receptor block the epitopes of both Abs, is there other potential reasons for my problem?
Thanks a lot!
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