I am trying to do chromatin immunoprecipitation with a GFP tagged transcription factor. I have tried a commercial anti-GFP antibody and one from another lab. I am verifying that my IP works via western blot, where I am blotting with an anti-GFP Ab that is different from the one I IP with, and have not been able to detect my protein::GFP from the ChIPed samples. I can definitely detect my protein::GFP via western when I run boiled worm lysate, so I am concerned that the problem lies within the antibody I am using for ChIP. Any good recommendations for anti-GFP antibodies, especially in regards to ChIP? My end goal is to do qPCR of potential targets. Thanks in advance!!
A quick look at some results from a Google query finds several papers in which people did ChIP on C. elegans using polyclonal anti-GFP antibodies (rabbit, from Clontech: 1,2; or goat, from the Hyman lab: 3).
Thanks Hillel. I have tried the goat anti-GFP from the Hyman lab with no luck so far. I might try that one from Clontech. I have also read about the Abcam AB290- anyone have comments about using this for ChIP?
I am wondering if part of my problem is also that I am not growing up enough worms to start with. After talking with some labs who have done ChIP, they grow worms in liters of liquid culture to get enough starting material. I have been doing a bleach prep from 40 150mm plates and then harvesting L2s for the ChIP. Any suggestions from anyone about this? My gene is only expressed in about 5% of cells and it’s an extrachromosomal array that’s reasonably penetrant. Thanks!