CHIP - only negative results! - (Nov/04/2008 )
Hello
I am doing CHIP assays using H3K27me3 and acH3 antibodies. I did PCR for two genes (the one gene is expressed and the other is silenced) for two different plant varieties, but I get no band in my samples. Because I have just started the CHIP assays, I don't know if there is a problem with the technique or if my results are ok. Do you think is possible not to find any of these two modifications? I examined one promoter region and one coding region for each gene. I mean I expected to find H3K27me3 for the inactive gene and acH3 for the active. Does anyone has any similar result?
Thanks very much in advance
Hi there 
We had a fair bit of bother finding good primers to determine if our H3K27me3 ChIP was working. At first we tried heaps of primers that were published in papers, but unfortunately none of the areas were trimethylated in our samples!!! We now have a reliable control region (only after doing 10+ chip-chips!!).
Good luck!
Clare
I am doing CHIP assays using H3K27me3 and acH3 antibodies. I did PCR for two genes (the one gene is expressed and the other is silenced) for two different plant varieties, but I get no band in my samples. Because I have just started the CHIP assays, I don't know if there is a problem with the technique or if my results are ok. Do you think is possible not to find any of these two modifications? I examined one promoter region and one coding region for each gene. I mean I expected to find H3K27me3 for the inactive gene and acH3 for the active. Does anyone has any similar result?
Thanks very much in advance
Thanks Clare. I believe that we can't afford so many CHIP assays 
Hmm...has someone else already done a successful ChIP with your plants in the lab (even if with another antibody)? Perhaps you need to design primers to different areas of the gene. Eg: alot of our H3K9 (acetylation) is within the first intron. Not sure what plants are like though!
Clare
No, nobody else does CHIP in my lab, and also there are no published results for barley regarding these two antibodies.
Hmm, well that sucks
Perhaps design some more primers?I am doing CHIP assays using H3K27me3 and acH3 antibodies. I did PCR for two genes (the one gene is expressed and the other is silenced) for two different plant varieties, but I get no band in my samples. Because I have just started the CHIP assays, I don't know if there is a problem with the technique or if my results are ok. Do you think is possible not to find any of these two modifications? I examined one promoter region and one coding region for each gene. I mean I expected to find H3K27me3 for the inactive gene and acH3 for the active. Does anyone has any similar result?
Thanks very much in advance
To test ChIP with the acetylated H3 antibody try designing a primer to about 100-300bp downstream of the transcription start site. We've had more luck detecting it here than trying to find it in a region that may or may not be the promoter. Also, for testing to see if the method is working for you, the 4H8 monoclonal for the Pol II CTD works really well (and since it works in mammals and yeast it'll probably work in plants) and will have a very high signal just downstream of the transcription start site.