Thank you, Paul.

The issue with the salmon DNA-blocked beads is that I didn't get any IP - so with these beads too high background is not the problem.

I guess the best idea is pre-incubate your proteins with protein A (PAG) for two hours at 4 C, centrifuge (8 000 g X10 min) and then use this supernadant for starting your IP. I never block PAG and I do not have problems with background or inespecific interactions. I can share my protocol if you want. Cheers!

Hi Maria,

Thanks for your advice! I actually did pre-clearing in the experiments, even in the ones with beads pre-blocked with DNA. Do you think DNA-preblocked beads could actually bind proteins during the preclearing, so that I loose them before the actual IP?

Cheers!

Yes, I think that could happen, but did you try to IP your protein and run a WB against the same protein... maybe it is a good idea do it with and without block the pearls. The pre-clearing is always necesary, because if you skip that part, you will have a lot of background. If you can see your protein on the WB, maybe the interaction between your protein and the other protein that you are looking for is very weak. Then a good idea is to decrease salt concentration (NACl) during the washes. I use for straight interactions 150 mM NaCl... :) :)

Thanks for your suggestions Maria, I'll try it ASAP. :)

Preclear your cell lysate with protein beads only,,, such that any protein binding to beads non specifically will be eluted out. the use the pre cleared cell lysate for IP experiment (with new beads),,,, good luck