Problematic results with ChIP - need help troubleshooting - (Oct/17/2005 )
I have been trying ChIP for eight months now without success using a commerical antibody (designed for ChIP) on rat pituitary cells. I was able to performed ChIP at another lab, however, in my lab I have never had any success. I started with the Upstate ChIP kit and followed the protocol exactly. I optimized sonication and I have never had troubles pulling out good quality Pre-IP DNA. Unfortunately, I have never pulled down any post-IP DNA. I also tried other protocols without success. So, I thought I would give this group a try!
First, I am wondering if anyone has ever had any problems isolating IP DNA through ChIP. I am looking for suggestions on things to try.
Second, I have recently noticed that there is a white precipitate towards the end of the ChIP procedure (much greater quantity than expected for IP DNA). I suspected it might be that the NaCl concentration was too high, but several washes of 70% EtOH has little effect. Column cleaning also resulted in little change to the sample. I was wondering if anyone has experienced this before and/or can suggest a possible solution.
Any help/suggestions would be greatly appreciated.
Thanks in advance,
Hi Chipper, some things to consider:
1. The commerical antibody, is the antibody directed to the rat isoform of the antigen? Otherwise, does it cross react with the rat isoform? Even though it has been done by the supplier, have you performed a western blot on whole lysate preps of your rat cells to determine if indeed the antibody is recognising your protein of interest?
2. Cross-linking, you may need to optimise cross linking times as over cross linking has a tendency to mask your epitope and the antibody will not IP your chromatin efficeintly, I would suggest less cross linking time if you are not seeing any IP happening.
3. are you using glycogen for your DNA precipitation? if so, this could be what you are seeing.
Thanks for the quick reply. The antibody is created for rat, however, I have not tried a western blot. I am going to try one and see if the antibody is actually hybridizing to the DNA. As for the crosslinking, I have already performed optimization experiments and we are working at the optimum. As for the glycogen, I think you may be right. There is glycogen in the sample and it had not occurred to me before then that it myight be this. I will have to try a few things and see if I can make any progress.
Thanks again for your help,
glad to be of help :-)
glycogen should not affect downstream applications of your IP DNA.....hasn't in the past for me anyway.
good luck with it!