ChIP fragment issues - (Sep/29/2010 )
Hi All,
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????
Thanks much.
samira_c on Wed Sep 29 22:04:39 2010 said:
Hi All,
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????
Thanks much.
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...
MM
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.
Mighty Mouse on Thu Sep 30 03:19:35 2010 said:
samira_c on Wed Sep 29 22:04:39 2010 said:
Hi All,
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????
Thanks much.
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...
MM
This might help (at least worth trying, right?) as most of the commercial protocols assume you are getting cells from cell culture I believe. After my washes following FA+gly I dounce homogenize in a glass tissue grinder (Wheaton) in a cell lysis buffer (10 mM Tris-HCl (pH 8.1), 10 mM NaCl, 1.5 mM MgCl2, 0.5 % Igepal-CA630), spin down at 14kg, remove supernatent and then re-suspend in nuclear lysis buffer, incubate on ice 10 min then store at -80C....hope that helps...
MM
I dont think a nuclear step is the first thing you should focus on. I also use a Branson 450 but the digital version. I do 8 rounds of 10 second pulse at 20% power output with 3 minutes rest on ice between rounds. I found that a number of things are VERY important with the Branson. First, make sure you have the right tip. You need the tip which best fits the volumes that you are using. For most of us chippers, that is the stepped micro-tip. Second, make sure you optimize the volume and amount of tissue. For example, with the micro-tip, you really should not go above 500 ul or below 200 ul and if you have too much material in there, sonication is inefficient. Lastly, you have to very carefully work out the conditions of sonication. If the power is too high, nothing will really happen even if it looks like it is and if you have the wrong tip, this is magnified. I personally think these are some things to get going on. I would consider working with a cell culture positive control sample also since your brain method is a little unusual compared the standard cell culture protocol.
Hi all, thanks very much for your tips and help. Will especially double check which tip for the sonifier I have, and will try some other conditions... hopefully will be able to report back with good news!