I have been working on a ChIP for a while, and am not sure what I should be expecting for % Input values of ChIP with specific antibody vs. no antibody or IgG. My protein of interest is not a fully established DNA binding protein in vitro (beyond biochemical work), so this is largely a discovery project to determine where my protein is binding (ultimately doing ChIP-Seq). Compared to IgG or no antibody, I definitely see some degree of enrichment with my antibody chip, but it's not astounding, and so I'm not sure how to interpret my data. The following is an example of qPCR that I did with a predicted target (based on a previous Ab shift assay with my protein of interest). Any input would be appreciated.
The percent input and fold enrichment you get are not too bad compared to a lot of publications on transcription factors. However, I can suggest a few things. 1) Definitely show IgG rather than no antibody control if you publish the data. It's also very important that the control be as similar to the specific antibody as possible (same species, sera vs. purified, etc) 2) The Ct for your no antibody control seems low (i.e. high binding). If you improve the washing or blocking steps, the fold enrichment might look better 3) Reproducibility is critical. Do you get very similar results if you do the experiment several times? 4) I think it's essential to do PCR with a target sequence that your protein should not bind. If there is similar enrichment with an irrelevant sequence, the result might be more of an artifact. 5) It may helpful to do a positive control ChIP for a transcription factor to get an idea of how good a result you can get with your methods (a histone control could also be used, but it should give a lot higher binding than you would likely get for your protein). Good luck!
The percent input and fold enrichment you get are not too bad compared to a lot of publications on transcription factors. However, I can suggest a few things. 1) Definitely show IgG rather than no antibody control if you publish the data. It's also very important that the control be as similar to the specific antibody as possible (same species, sera vs. purified, etc) 2) The Ct for your no antibody control seems low (i.e. high binding). If you improve the washing or blocking steps, the fold enrichment might look better 3) Reproducibility is critical. Do you get very similar results if you do the experiment several times? 4) I think it's essential to do PCR with a target sequence that your protein should not bind. If there is similar enrichment with an irrelevant sequence, the result might be more of an artifact. 5) It may helpful to do a positive control ChIP for a transcription factor to get an idea of how good a result you can get with your methods (a histone control could also be used, but it should give a lot higher binding than you would likely get for your protein). Good luck!
Hi Ryan,
I agree with Michael. Your % Input value(s) for the sample are in an acceptable range. No Ab ChIP should definitely yield higher Ct values (around 38 or later or not detectable at all after 40 cycles / how many cycles do you do for your qPCR?). I also think that IgG is a better control than no Ab at all (and in my experience it gets even cleaner); nonetheless, the best control is a negative control region where your protein doesn't bind. I know the struggle to find such regions, since I also didn't know at first where to expect binding and where not. If you're working in mouse, I could give you some primer sequences that you could test, also for human I should have a handful. Also along the lines of Michael, I would suggest an established positive control, i.e. polII ChIP or histone (any mark or even pan), just to make sure the ChIP protocol works in your hands. This also gives you confidence to trust your %Input values/ChIPs for your protein of interest.
Feel free to contact me, in case you need primer sequences or have any more questions.
Best,
Kevin
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