I have started doing ChIP a few months ago and am trying to optimise some conditions. I have a few questions/concerns:
1) How necessary is the NaHCO3 in the elution buffer? I am following a protocol used by a previous student, and adapted from a "micro ChIP" paper, and in this protocol the elution/reverse cross-linking/proteinase K treatment is done all at once by shaking at 1300rpm, 68 degrees, for 2 hours. However this elution buffer consists of SDS, NaCl, Tris and EDTA - no NaHCO3.
2) What is an acceptable amount of input DNA to get back with antibody, and what CTs from qPCR are okay? The most recent ChIP I did had these numbers:
Input (200 ul starting material, 22.2% of IP) concentration: 40 ng/ul in 40 ul (total 1.6 ug DNA). Running 2 ul of this undiluted has a CT of 19 for target gene.
Antibody (900 ul starting material): CT of ~26 for target gene, CT of ~30 for control region
IgG control (900 ul starting material): CT of ~31 for target gene, CT of ~31 for control region
So I have an enrichment of 64-fold over IgG for my target gene, but an enrichment of 2-fold over IgG for my control region. I assume this normalises to ~36-fold enrichment? Furthermore, when I calculate the % of input that I get with antibody, it is about 0.3% - is this okay, or very low??
3) I've been trying a ChIP with a different antibody (for diff protein). This time, I get:
Antibody: CT of ~28 for target gene, CT of ~28 for control region
IgG control: CT of ~31 for target gene, CT of ~31 for control region
So just looking at the target gene, it looks like I have ~8-fold enrichment over IgG, however the enrichment is also there for the control region. What would you suggest to try to overcome this? Would more washing help remove the non-specific binding, more antibody/less antibody? I currently do not have the LiCl wash as part of my protocol, which seems to be working okay for the previous antibody, but would doing this possibly help? I am already using magnetic beads which are blocked in BSA.
Any input would be greatly appreciated, thanks!!!