ChIP assay results interpretations - (Oct/31/2006 )
I am new in this field and thank you to all of you I progress in my experiments with ChIP.
I am using GADPH amplification for IP with RNA pol II as a positive control and for negative control the primers suggested by active motive.
I have one question for the interpretation of my results:
I expect
1) the input DNA PCR (sheared chromatin with out IP with an specific antibody) to be higher always that the sample IP;
2) PCR DNA band with the negative control primers should be always the same with and without IP.
3) PCR DNA band with possitive control and test samples should be higher in the samples IP that in the ones.
Is this true?
Thank you 
I am using GADPH amplification for IP with RNA pol II as a positive control and for negative control the primers suggested by active motive.
I have one question for the interpretation of my results:
I expect
1) the input DNA PCR (sheared chromatin with out IP with an specific antibody) to be higher always that the sample IP;
2) PCR DNA band with the negative control primers should be always the same with and without IP.
3) PCR DNA band with possitive control and test samples should be higher in the samples IP that in the ones.
Is this true?
Thank you
1) Yes, the input sample should have a higher signal than the IP ... unless you dilute it too much. As long as you use, for the input sample, about 1/100 or more of the material that you use for each IP, the value should be higher.
2) Yes, the signal you get with the negative control primers should be approximately equal for both the IP and the no antibody control IP (or IP done with total IgG or epitope blocked antibody).
3) Yes, the signal for GAPDH should be quite a bit higher in your IP than in your no antibody control IP (assuming your cells haven't shut down glycolysis).
Hope that helps.
KPDE,
Thank you for your answer. 
Do you think it is necesary to sequence PCR fragment to be sure the promoter region amplified was the one in the PCR?
By the way I am ususing tissue and I started with aproximately the same amount, so I am assuming to have same number of cells.
I am comparing tissue samples from disease patients and from normal subjects. How I can be sure that the difference between different samples is due to binding of these proteins to DNA and is not just a false positive?
Thank you and have a great day
Thank you for your answer.
Do you think it is necesary to sequence PCR fragment to be sure the promoter region amplified was the one in the PCR?
By the way I am ususing tissue and I started with aproximately the same amount, so I am assuming to have same number of cells.
I am comparing tissue samples from disease patients and from normal subjects. How I can be sure that the difference between different samples is due to binding of these proteins to DNA and is not just a false positive?
Thank you and have a great day
The easiest way is to normalize using your input DNA. Just express the result as the ratio of your IP over your input.