losing the signals in ChIP - (Dec/22/2008 )
Hi everyone,
I've read the postings in the forum and found them quite helpful. I think I've seen two more people having the same problem. When I started ChIPping, It took me a couple of months to optimize but after that things were working ok. Then after a few months, suddenly I've started to lose the enrichments that I was getting before. And since then, I've tried almost everything(new solutions, changing starting OD, different sonication power and settings, new beads and FA, different washing conditions and crosslinking times, different protocols), but could not make it work again. After much effort, I could get some enrichment again for my control RNA15-TAP, which is a 3' UTR binding protein. But it's a very narrow spectrum of optimal conditions and does not work for galactose conditions.
my culture is 200 ml and OD is around 0.5-0.6.
I'm using vortex to lyse yeast cells, what's the best way to lyse yeast cells using a vortex? I'm using 200 ul beads + 300 ul yeast suspension + Prot. inh.s and 1 hour vortex at 4 C.
and some protocols use WCE and some discard sup before sonication. which one is better?
I'm using using fisher sonic dismembrator 100, does anyone have optimized settings for it? I'm either using setting 3 for 1 ml or setting 7 for 2 ml for 12 X 10''.
do you do something different when you grow cells in galactose and ChIP on Gal genes?
sorry for the long posting, I'd appreciate any suggestion.
Does anyone have any idea what can be a common reason for such a situation?
I've read the postings in the forum and found them quite helpful. I think I've seen two more people having the same problem. When I started ChIPping, It took me a couple of months to optimize but after that things were working ok. Then after a few months, suddenly I've started to lose the enrichments that I was getting before. And since then, I've tried almost everything(new solutions, changing starting OD, different sonication power and settings, new beads and FA, different washing conditions and crosslinking times, different protocols), but could not make it work again. After much effort, I could get some enrichment again for my control RNA15-TAP, which is a 3' UTR binding protein. But it's a very narrow spectrum of optimal conditions and does not work for galactose conditions.
my culture is 200 ml and OD is around 0.5-0.6.
I'm using vortex to lyse yeast cells, what's the best way to lyse yeast cells using a vortex? I'm using 200 ul beads + 300 ul yeast suspension + Prot. inh.s and 1 hour vortex at 4 C.
and some protocols use WCE and some discard sup before sonication. which one is better?
I'm using using fisher sonic dismembrator 100, does anyone have optimized settings for it? I'm either using setting 3 for 1 ml or setting 7 for 2 ml for 12 X 10''.
do you do something different when you grow cells in galactose and ChIP on Gal genes?
sorry for the long posting, I'd appreciate any suggestion.
Does anyone have any idea what can be a common reason for such a situation?
I probably sound like a broken record but have you checked to see if you are using a different lot number for your antibody (or a different preparation is you are making it yourself) now then when ChIP was working better for you? Different lots can have different concentrations even if there is no indication on the vial (e.g. Cell Signaling). If they're polyclonal the different lots can come from different animals.
Thanks for the reply,
I use TAP-tagged proteins, so I directly use IgG beads. Two sources of IgG beads I use: one is from
GE healthcare and the other is from Sigma. Sigma one give ssubstantially high background(several times higher signal for control region over some target regions). This is like that even I use only 2 ul of beads, probably it needs more stringent washes. GE beads work ok for some genes, I think it's more about the region I try to IP, because for other genes I could get results.
Interesting thing about my regions is: I get different amounts of DNA for different regions, this is especially true for
Gal region. twice more DNA for control region compared to my target region around Gal gene. This probably shows that
I either solubilize less or sonicate less efficiently the chhromatin for that specific region. I'm planning to incubate like one hour in FA-lysis buffer after cell disruption and wash and before sonication. and increase sonication time. I'm still trying to solve the problem. open for any suggestions for ChIPping Gal genes in yeast.
Thanks