Dear all
I would like to ask some questions about my current ChIP experiment. I bought the ChIP assay kit from Cell Signaling Technology (#9002 SimpleChIP @ Enzymatic Chromatin IP kit with Agarose Beads) to do the experiment, and want to know the association of transcription factors and a promoter in our study.
I have performed twice ChIP assays , and I think I still not catch a well condition of nuclear lysis of my cells by sonication after Micrococcal Nuclease digestion (following protocol's recommendation that after S7 nuclease digestion, the fragmentated chromatin will vary in a range of 150 bp - 900 bp), cuz I ran agarose gel of digested chromatin, the bands were so fuzzy and unclear, and the DNA concentration is so low after OD260 measurement. The chromatin prep. was prepared from about 2x107 cells, and I think the conc. should not be low as this way. Hence, I think the problem may from the low efficiency of nuclear lysis after sonication.
In spite of low chromatin concentration, I used 5 ug of chromatin DNA in each IP set. , however, I got amplification signals using RPL30 primers and other primers from IgG negative ChIP sample !!!
So, I'm writing to ask you kindly guys if there are any recommendations in your experiences? How do I control my sonication condition to lysis nuclear membrane, and how I know whether it is completely lysed or not ? And how about false-positive results of IgG control, what should I do ?
Mason
A couple of suggestions (if you are not doing this):
Sonicate with chromatin sample in eppendorf on ice. The sonication probe will occupy most of the eppendorf, which is OK as long as the sample is on ice. Sonicate for 15 secs, then turn switch off, sonicate for 15 secs again, back and forth. Can remove samples as time points, run on agarose gel. The chromatin will largely be a smear, if you get smaller and smaller sizes than your sonication is working. Stop when average size is around 300-500 bp.
You are going to get some negative control amplification, The point is that your region of interest (promoter) is amplifying with fewer cycles (it maybe just 2-3 cycles) then the negative control (Intron in gene).
Some refs;
My paper with a detailed ChiP protocol, which should work. Note all the negative controls I did ( compare TF binding to promoter versus Intron, etc):
http://www.ncbi.nlm.nih.gov/pubmed/22467658
This is an outstanding methodology paper that helped me a lot:
http://www.ncbi.nlm.nih.gov/pubmed/17892552
Steve Presnell
HI,
quite a bunch of questions you have. I try to answer the problems I was facing,too.
First of all an agarose gell of sheared chromatin is rather a smear with a peak like increase for a certain size range. For me it came out normally to have this "peak" at 400-600 bp and my maximum was around 900 bp. I use a covaris sonicator for shearing so no enzymatic digest. This step also opens my cells and lysis seems to be quite complete. HOw do you crosslink your chromatin? I normally do 15' 1% FA @ Room temp. Instead of DNA amount I normalized on my cell numbers before crosslinking. I normally take 5 millions of cells/ condition. If you use agarose beads you may think about preclearing your lysate and blokcing the beads with unspecific DNA. This could help your with xour background problem. At last hte point with your low DNA concentrations. How do you extract your DNA ( I use DNA cleanup from macherey nagel) and why do you think it is too less?
Hope this helps a bit
regards
Sven
About false-positive results of IgG control, I guess it's unavoidable, when your expected signal is much higher than this, then the IgG signal can be accepted. And use another negative control such as some exon sequence which TF do not bind with.
The real problem here may be that at which time point should you collect the cells after treatment? Hope this pdf can give you some hints about this.
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