ChIP problem - using cells as a negative control
Posted 05 July 2013 - 01:01 PM
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!
Posted 10 July 2013 - 03:56 PM
Posted 11 July 2013 - 01:30 AM
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?
Edited by uhsiracu, 11 July 2013 - 07:09 AM.
Posted 22 July 2013 - 08:07 AM
Posted 22 July 2013 - 08:12 AM
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?
Edited by uhsiracu, 22 July 2013 - 08:30 AM.
Posted 08 October 2013 - 11:02 AM
I have similar problem. I got enrichment at non-binding area.
Posted 27 November 2014 - 10:27 PM
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