Agarose / salmon sperm slurry - from upstate (Sep/05/2005 )
I have recently tried to do a ChIP assay following someone elses protocol, including the same antibody they used. The only thing I did differently was use the Protein g agarose we have in the lab rather than the Agarose/salmon sperm slurry from upstate.
My ChIP didn't work - could it have been because of this difference? Should I convince my supervisor to buy some from upstate?
What do you mean by "didn't work" ? no bands or no enrichment?
I got a band in my total input PCR, but not from my +Ab sample. So... no enrichment?
Do you know if the agarose/salmon sperm slurry was also protein G?
As long as your Ab binds to the bead you should be okay, but some antibodies bind protein G but not protein A and vice versa...
Another possibility is that you have pulled down something, you just can't detect it.... many people do two rounds of PCR for ChIP you could do dilutions of your input to see how sensitive your PCR is, you should definately see bands at a minimum of 0.1% of the input or else your IP probably didn't work. (These assays can be very inefficient, but you should at least get 0.1% pulled down with the antibody, I usually get around 1%.)
The whole reason for buying the agarose salmon sperm slurry is to reduce background. If you aren't having background problems, then it shouldn't make a difference if you use the upstate slurry or not. I wouldn't worry about buying unless you need to reduce background...
Thanks for that - I was using ptn G. But I will definately try another round of PCR. fingers crossed!!!
I think that two rounds of PCR is excessive. If you cant see it by one round its probably not there. You shouldnt have to go to the level of single copy. I think if you do the result is not very believable.
What is the antibody? Mouse monoclonals bind well to Protein G and rabbit polys to Protein A.
I think that you may have pulled down nothing.