About the control choice in a ChIP-experiment.

Question

[ghostforever.shi]

I am going to use H3K4me3 to test the H9 ES cells. One of my friends suggested me to use H3 antibody as a control, but he didn't explain the reason. I wonder why I am supposed to use H3 as a control and what is the difference between using IgG and H3 as controls. Or whether I should use them both if necessary?

Answer 1

[Devon Ryan]

IgG is rarely used as a control these days because it turns out to be very problematic to use (due to getting very very very low amounts of DNA from the IP). In general, it's best to use a straight input sample (i.e., something that wasn't ChIPed) or H3. I'm not a huge fan of using another IP as a control, since then you're relying on multiple antibodies, possibly with different IP efficiencies. Having said that, if the H3K4me3 antibody isn't completely specific to the modification, then using H3 as an input will certainly help to remove non-specific H3 peaks.

[ghostforever.shi]

Oh, I see. Much appreciate!