IP looked like it worked - Mass Spec says different - (Jun/16/2011 )

I was looking to see if someone could help.

I've been trying to perform Immunoprecipitations recently. I've been using a standard Pierce co-ip kit. And using the column making instructions they gave with it (http://www.piercenet.com/instructions/2162181.pdf)

I have 3 columns for running lysates through.
One has my antibody bound to the resin.
A second column is a control with antibody quenching buffer added.
Then a third column is a control with an inactivated resin.

I've tried optimizing, by adding varied amounts of lysate.
Eventually i got to where i thought it was working.

I was running western blots to confirm the sample worked.
I'd run the eluted protein samples, and the lysate that was flowing through, and some of the washes also.
A band only appeared on the eluted protein where my antibody was added.

So i sent this off, with the control elutions for comparison, to be analysed on a mass spectrometer.

However, the results showed back pretty much the same proteins across all 3 samples.
I think it might be non-specific proteins may be getting in my way.

I was wondering if anyone had any suggestions of what i could try to remedy this whole problem?
Thanks.

Have you done westerns on whole cell lysate using the same antibody you mentioned? If so, did you see more than one band?

MS is very sensitive, so you could be getting some binding coming through in your quenched sample. Could you compete the band out from your IP with a peptide against the antibody epitope?

Are you seeing keratins by any chance?

bob1 on Fri Jun 17 00:03:31 2011 said:

The peptide thing is a test to see if your antibody is specific buy competition. Antibodies are raised against peptides, and the recognition site of an antibody on a peptide is called the epitope. If you know what the epitope for your antibody is, you can then get the peptide and pre-bind it to the antibody. IF you then use this pre-bound antibody on a western or IP, you shouldn't see any band, so you know your results are specific (i.e. you aren't looking at a non-specific band). As western blotting is not a very sensitive technique, this competition may be a better control for your experiments than quenching the antibody.

The keratins are a sign that your mass spec is working properly - these are from shed skin cellls that are floating around in the lab - just to give you some idea of the sensitivity of the mass spec.