low assay sensitivity - (Nov/03/2008 )
Hi all,
I work in a CHO system and would like to quantitate (via ChIP) how many copies of a transfected gene are located in open / transcriptionally active chromatin versus closed / transcriptionally silenced chromatin. In order to do so, I'm using the following internal controls: a housekeeping gene to ID the open chromatin fractions, and a developmentally silenced gene for the closed chromatin fractions. I'm using ChIPing grade antibodies against acetylated histone H3 (for pulldown of open chromatin) and antibodies against monomethyl and dimethyl histone H3 (for pulldown of closed chromatin). I'm able to get a nice signal for my open chromatin controls, but I've been unable to get good quantifiable signals for my closed chromatin controls. I've upped the amount of cells I use per IP, and I've upped the amount of antibody per IP - NO LUCK!
Does anybody have any suggestions?
I work in a CHO system and would like to quantitate (via ChIP) how many copies of a transfected gene are located in open / transcriptionally active chromatin versus closed / transcriptionally silenced chromatin. In order to do so, I'm using the following internal controls: a housekeeping gene to ID the open chromatin fractions, and a developmentally silenced gene for the closed chromatin fractions. I'm using ChIPing grade antibodies against acetylated histone H3 (for pulldown of open chromatin) and antibodies against monomethyl and dimethyl histone H3 (for pulldown of closed chromatin). I'm able to get a nice signal for my open chromatin controls, but I've been unable to get good quantifiable signals for my closed chromatin controls. I've upped the amount of cells I use per IP, and I've upped the amount of antibody per IP - NO LUCK!
Does anybody have any suggestions?
Which antibodies are you using for methylated H3 (i.e. what company made them and what residue are they for)? Monomethylated and dimethylated H3 is pretty non-descriptive. I'm guessing you know for sure that the silenced region you are looking at has the modification which you are trying to pull down, in CHO cells.
So, are you not getting signal at all in the silenced region or are you just getting a signal which is similar to background (mock). If it's the latter, try using less chromatin and the same amount of antibody. If it's the former, there could be a lot of problems. Have you run positive controls to make sure that the antibodies work (I'm sure you've run the relevant controls to make sure your primers work at the silenced region)?
We've found that silenced areas of the genome are more sensitive to proteinase K treatment (i.e. less treatment give less signal). You might try incubating with proteinase K for longer. Also, if you are shearing your DNA with a sonicator, different areas of the genome are sheared with differing efficiency. In general, the more open areas are sheared more efficiently. If you are seeing a fair amount of high molecular weight DNA after shearing it's possible that the area you are interested in is primarily in this un-sheared smear, and is not efficiently pulled down.
The most likely problems are the simplest so I would start by making sure that the antibodies work in your assay using primers for a positive control region known to have H3 methylated at the residue you're looking at.
Good luck.