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ChIP problem - using cells as a negative control - (Jul/05/2013 )

Hi everyone,
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!

-uhsiracu-

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

-chabraha-

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?

-uhsiracu-

Any suggestions??

-uhsiracu-

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

-chabraha-

Hi Chabraha,
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?

-uhsiracu-

I have similar problem. I got enrichment at non-binding area. 

-mc1061dm1-