Sudden, Non specific pulldown in Co-IP's - Why? (Nov/28/2006 )
I identified several protein interactions by doing mass spectrometry of unique gel bands run on SDS page after co-immunoprecipitation. Problem is recently, in trying to verify them using western blotting, the proteins started being precipitated with my IgG controls, which is infuriating . I validated a couple of these proteins by western blot a week earlier and it appeared to be a specific co-precipitation (in the FLAG pulldown but not the IgG control).
I am using the same buffers, and have tried remaking them (with and without 50mM NaCl to increase specificity) but the problem persists. I am also using the same protocol, that I originally used for the identifications of the interactions, so I am unsure why this is happening. Does anyone have any pointers for reducing background in co-IP experiments in general???
I use a high protein concentration (could be a problem?, but inside the cell is VERY high too)
try to limit bead volume so as not to pull down aggregating proteins
use confluent cells so as to get a lot of protein from 3 10cm plates per group
use HEPES/acetate buffer
0.5% NP40, 25 mM HEPES pH7.35, 115mM K Acetate, 5mM MgCl2, 1mM Na3VO4, and complete mini tablet.
ANy ideas would be greatly appreciated
PS...I am seeing stringy things in my beads after I mix them with the lysate so I am going to also try recoupling fresh beads to antibodies and try the IP's with freshly coupled beads. Any Other ideas or things to try optimizing????
I don't quite understand your IP method...do you do plain IP as in lysate+Ab then + beads?
anyway...I can only think of preclearing the lysate with the IgG first
then the precleared lysate IP with antiFLAG
or, could it be possible that your sample loading leaked to the neighboring well?
(was it a one time thing?)
hope you have it solved already.