Problems using a peptide antibody - Troubleshooting (Dec/03/2008 )
Hi,
I'm trying to optimize a ChIP protocol using a peptide antibody. As far as I know, ChIP has not successfully been completed for this protein. We've tried to perform ChIP at several different potential binding sites of known targets and none of them seem to work. I can get ChIP to work using positive controls. I'm concerned that we're having this problem because we're using a peptide antibody and may be having some sort of unmasking problems. Does anyone know of a way to address this issue?
Thank you very much.
-jp1229-
QUOTE (jp1229 @ Dec 3 2008, 09:06 AM)
Hi,
I'm trying to optimize a ChIP protocol using a peptide antibody. As far as I know, ChIP has not successfully been completed for this protein. We've tried to perform ChIP at several different potential binding sites of known targets and none of them seem to work. I can get ChIP to work using positive controls. I'm concerned that we're having this problem because we're using a peptide antibody and may be having some sort of unmasking problems. Does anyone know of a way to address this issue?
Thank you very much.
I'm trying to optimize a ChIP protocol using a peptide antibody. As far as I know, ChIP has not successfully been completed for this protein. We've tried to perform ChIP at several different potential binding sites of known targets and none of them seem to work. I can get ChIP to work using positive controls. I'm concerned that we're having this problem because we're using a peptide antibody and may be having some sort of unmasking problems. Does anyone know of a way to address this issue?
Thank you very much.
Very likely cause. The obvious suggestions would be to
1. Make antibody to another peptide seq in your protein. Design well, and pray.
2. Make antibody to protein, using a larger portion of your protein.
3. ChiP, in my experience, works best with polyclonal Abs raised against protein antigen.
There is still a chance that something else may be wrong. You can overexpress your protein in teh cell line and perform ChIP, perhaps the endogenous protein levels are too low.
-cellcounter-