I'm perfomring ChIP-seq now and I noticed that my protein of interest seems to come out from the washes, although some stayed until elution from western blot. However, I don't see people routinely doing WB to check ChIP quality. I wonder if other people have checked ChIP with western blot and should I optimize it so that I don't see my protein of interest during the washes?
We do this routinely to check for the efficiency of the IP. If you do not see your protein in the final elution then the IP did not work. You can take a fraction of the elution, add 2X SDS/PAGE buffer and 0.1M NaCl, boil for 5 minutes then reverse crosslinks at 65oC for 4 hours and run this sample on a western blot to check the enrichment for your protein. You can compare to the intiial input protein from chromatin and also to the depleted sample after removing the protein of interset with Dynabeads or Prot. A Agarose.
I am running the CHIP now, it cost me two months for troubleshooting before it work smoothly. I think it is reasonable to troubleshooting with WB, but I never tried WB to check ChIP quality. As Gregory Dressler described above, you need reverse-crosslink before run the samples, otherwise you cannot separate the crosslinked proteins-DNAs complex. After all, WB is not a best method to track you CHIP, you'd better try some other ways to checked ChIP quality.
It might be that your IP is sub-optimal, you can play with amount of Ab, beads and staring amount to make it more efficient.
Gregory Dressler Irina Panteleeva Shaohua Qi Thanks everyone for the insights. Now it seems like all of my protein of interest comes out in my washes, more specifically in 1st wash flow through. I wonder what lysis buffer composition you guys use. I have 0.1% SDS and 1% Triton X100. I think that either my protein of interest or the antibody is not binding to the beads. Also I'm doing a ChIP not just IP so I'm not sure if the lysis buffer needs to be milder.
Can you freeze at any step in this process? Can I freeze at -80C after boiling and then thaw and reverse crosslink?
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