Hi, thanks for the response.
So actually, my protocol (based on some previously published papers by authors using the same tissue region) does not contain a nuclear fractionation step. Actually, many of the protocols (ie : from activ motif, or abcam) don't mention isolating the nuclear fraction. So maybe this is the missing link...??
I currently mince/chop the brain tissue prior to crosslinking, after a couple of washes, homogenize in 1% SDS, sonicate in this same SDS lysis buffer for some indicated timepoints, and then add water, NaCl, and incubate overnight in proteinase K at 65 degrees, RNase A at 37 degrees for 30 minutes, and then boil at 95 degrees to reverse crosslinks.
So frustrating! Perhaps I will need to do nuclear fractionation.
I suspect this question must be asked all the time, however, after reading the archives and this forum, I am completely stumped. For the past three months, I have been trying to get ChIP working in my lab. We are using adult whole mouse brain tissue, and will later specifically use adult striatal brain tissue. We have tried starting with both 100mg and a lower amount , 30 mg, of tissue and we currently lyse in the the traditional 1% SDS buffer which has also been discussed on the boards. However, I never seem to get my ChIP fragments below 1kb (the 200bp to 1 kb range). When I take a picture after first running the gel (1% agarose, in 0.5x TBE buffer), it seems like the chromatin fragments might be in the appropriate range, but after running the gel out longer, it shows that most of the fragments are at 1.5 to 3kb. Please help... what am I missing????
Not altogether sure I have an answer for you as I'm not that familiar with using the wand sonicators, but presuming the settings on the sonicator are appropriate it looks to me like you either have too much crosslinking or inefficient sonication. 10 min of 1% FA at RT then the addition of glycerol shouldn't be too much crosslinking (I assume there is a cell lysis and nuclear fractionation step too?), so perhaps you are not re-suspending your nuclear fraction in the appropriate amount of nuclear lysis buffer? For what it's worth I also use mouse brain tissue, bilateral hippocampi, which I believe come to about 30mg of tissue. I re-suspend my nuclear pellet in 300 uL of a lysis buffer that contains 1% SDS prior to sonication with a BioRuptor...