In a protein IP from HeLa nuclear extracts I've recently used protein A agarose beads pre-blocked with BSA and salmon sperm DNA (which we use for ChIP). The problem is: I don't get any pulldown.
The reason for using these beads was that the background using magnetic beads blocked only with BSA seemed high.
Since I'm interested in nuclear proteins that potentially bind DNA I wanted to reduce background due to proteins sticking to DNA and beads.
Has anyone used DNA-blocked beads for protein immunoprecipitations successfully? Do I maybe have to use special buffers?
Thanks!
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... :) :)
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