hello everyone, i'm doing chip in insect cells. I use Richard A Young lab published paper in nature protocol in 2006 :"Chromatin immunoprecipitation and microarray-based analysis of protein location".
i tried to optimze sonication, and result i got like this(sonicaiton.png), whether i can continue to do chip or i need to optimize my sonicaiton?
Besides, i used H3K4me3 and H3K27me3 antibody（merck millipore） to do chip, i got a poor results, the final concentration is 0.1~0.3ng/ul(volume 50ul). Agilent result is also not ideal(below figure).
is it caused by sonication? or some other reason?
anybody can give me some suggestion?
thank you very much.
Your sonication looks fine with most of the DNA between 250-500 bp.
Dear Cheng,
Your sonication profile looks perfect, move on and test if your IP worked by ChIP-PCR. The low yield in the end can be improved by other means; i.e., using more cells, purified the IPed DNA with AMPure beads instead of column-based purifications etc.
Good luck!
Antonio.
Dear Cheng,
Your Sonication looks good! Outcome depends on the input material you have used and antibody efficiency. Try to use more material. And we use Cell Signaling antibodies (Rabbit Monoclonal) for these histone mark ChIPs. AMPure beads are good idea.
Good luck!
Shuchi
Your sonications profiles look fine for me, as said above, and the profile of precipitated DNA with H3K4me3 antibody as well, even if quantities seem to be low. Are the BioAnalyzer profiles performed with DNA high sensitivity chips? If not, it helps a lot with a good quantification before making libraries for ChIP-seq. For this sample (H3K4me3), you might even have enough material to make libraries (which quantity / concentration) to you have when you take the main peak (100-1000 bp) in BioAnalyzer analysis?
I am quite surprised with the H3K27me3 profile, though, is it performed the same way as the H3K4me3? Did you reverse cross-link your chromatin? Ampure XP beads can be quite tricky to use and I find them useless at this step (before making libraries), I rather use MinElute columns, followed by ethanol / salt precipitation if you need to concentrate your samples. As said, you can increase you yield by using better antibodies, improve your ChIP protocol, use more cells... Good luck!
Thank you all of you, I will use more cells and improve my protocol.
Best wishes
ChengDong
1) your sonication does look fine apparently, but is this DNA RNase treated before loading on a gel. because RNA is always in excess and usually masks the DNA smear in the same range sometimes. So if you clarify this, I can rest assured that the smear u r observing is a DNA smear in the correct range (not RNA smear).
2) for ChIP input perform your IP at a dilution of 50-100ug/ml of chromatin measured as A260 and preferable bind your antibody to beads before adding it to the input chromatin.