I am really in a fix with Chip assay.
I am doing ChiP with an antibody for a transcription factor (TF) using Upstate's Chip assay kit. The antibody I am using is the one used for Chip successfully and published from another lab. The difference between them and us is the cell used for the assay. They used a cell line (MEF) which expresses the TF uniformly, while we are using a mouse tissue, in which only ~3% cells of the tissue expresses the TF. So the population of the target cells used is very very small in our case.
I used about 20 million cells (~0.6 million target cells) per assay. To increase the chance to meet the antibody with the target TF_Chromatin complex, I incubated the antibody (2ug) with sheared chromatin solution in 500ul (100ul chromatin solution + 400ul Chip Dilution Buffer), which is twice as high concentration as original protocol, at 4C overnight. In the beginning, I sheared chromatin in 1% SDS so that the concentration of SDS in the solution was 0.2%. The results were very variable: sometimes I saw 4-8 fold difference between target sample (qPCR Ct value: ~31) and negative control samples (IgG and No antibody, Ct value: ~34), but sometimes there was no difference. Later, thanks to this web, I noticed bad effects of SDS for the Chip and used 0.5% SDS for the chromatin shearing (the size of sheared DNA was as good as before (~500bp)) to lower the concentration of SDS in the solution to 0.1%. This time, results became worse than before. All signals become lower: qPCR Ct values of the sample and negative control were around 34 or not detected. No significant signal/niose fold difference was seen.
As a positive control, I am doing Chip using an antibody for a different TF, which also expresses in the ~3% cells of the tissue. In this assay, I have positive results consistently in both 0.2% and 0.1% SDS containing Chip. So the cell number I applied to the Chip should be OK.
In my understanding, the latter results (no signal!) could be the real results unfortunatelly. Maybe because of the lower affinity of the antibody. Should I give up using this tissue? If there are some suggestions or advice to improve the assay, please let me know.
Thanks in advance for reading this long letter and cooperation to fix the problem.
Edited by ChipChimp, 15 April 2009 - 07:42 PM.