ChIP problem - using cells as a negative control - (Jul/05/2013 )
I'm new in the forum, so nice to meet you!
I'm doing chip experiment since few months but I'm encoutering troubles with the controls. I explain myself better: I'm working on human cells and if I compare my sample data and the noIg control data, the chip seems working... but if I include as a negative control, human cells that don't express the TF I'm studying, then I get strange results in term of % input and fold enrichment: for example, I obtain more enrichment in my negative control cells than in the cells that express the TF! Does anyone have some suggestions or hypothesis? To be clearer, I didn't do KO of the TF in the cells that I use for negative control: they are just cells that don't express the TF.
Thanks in advance!
What cells are these? You need to compare a negative control DNA region between the samples........not just an IgG IP control.............also you might be using too much antibody
Hi Chabraha, first thanks for your reply. The cells I'm using as a negative control are Th1. I'm already comparing a negative control DNA region where I know my TF isn't binding and in fact there I have an enrichment in % input of 0.2 for example, while in my target region where my TF should bind, I obtain something around 1.4 %input. I'm using 1x10^6 cells and 2ug of monoclonal antibody. The point is that I know that these Th1 don't express the TF, so I should not see enrichment in the samples containing them, or at least I should see something comparable to my no-ab control: is that correct?
EDIT: I've also another problem: I realized that I have low background (around 0.1%) in region used as a negative control, but higher background (from 0.5 to 1%) in regions that are bound by my TF. Do you have an explanation? What I'm doing wrong?
Sounds like too much antibody or your incubation time with the antibody could be cut down substantially.............try the IP step for 2-4 hours................If your truly sure your TF is not expressed in these cells (at the RNA level) then sometimes I found that monoclonal antibodies can have a propensity to IP non-specifically in the absence of epitope..............especially at high concnetrations
thanka again for the reply. I just use 2ug per IP, but I will try with 1ug decreasing the incubation time (I'm making an overnight) and see what's going on. Yes, I'm sure that these cells that I use as negative control don't have mRNA of my TF. I'll keep you in touch!
EDIT: How can you explain the high pulldown in the no-ab sample?
I have similar problem. I got enrichment at non-binding area.
I have the same problem doing RNA Polymerase II-Ser2P Chip which I have higher enrichment in the intergenic regions as compared to positive controls (gene body of Gapdh and Actb). I have tried reducing time of incubation and the amount of the antibody but it didn't help