I’m conducting an immunoprecipitation (IP) experiment where I pull down samples using a specific antibody and subsequently perform a Western blot, probing the membrane with another antibody to detect a different protein. Interestingly, I’m observing signals in my control samples (IgG, beads only), which I suspect are due to endogenous expression of the other protein.
Has anyone encountered similar issues with endogenous protein detection in control samples?
Any insights or recommendations would be greatly appreciated.
Thank you!
with IgG only (no antibody) beads you should not have any signal (it is your negative control for co-IP) unless one of your proteins sticks to the beads (or with protein G but not likely if it is not an antibody) . Maybe you can eliminate the background by pre-incubating your lysates with the beads (sephadex not coupled to protG) or preincubate the beads with BSA...
Didier Poncet, thanks for your suggestion, I will try them. I am not getting any signal in my controls for my target protein (the one I am pulling down). But I am probing the membrane for another protein, and that one is being expressed in my control samples as well. Hence I was thinking that if my beads are somehow cross-reacting with that protein.
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