I want to precipitate the chromatin by following antibodies to know what else protein is binding to the site of interest. I have one protocol for Chromatin IP..but by following that I am getting very less DNA like 17 ng/ul some times 5, 1 and once I got good concentration like 72 ng/ul (50 ul).
I want to know about an effective method for Chromatin precipitation ?....If Somebody having experience with chromatin enrichment then share your view..
Thanks In Advance
What antibodies are you using for the Chromatin immunoprecipitation? It could be the antibodies are contaminated with the very same proteins that you hope to have them bind and precipitate.
Antibody I did not check but antibodies are from Active Motif and ChIP grade....
I would Like to know, what should be the optimum conec. of DNA after ChIP
I have never had an optimum concentration that I am expecting to get. It all depends on the amount of protein found on the chromatin that you are studying and the amount of pure antibodies you are using. Just remember, because it says "ChIP grade" does not mean it is absolutely pure of any antigen contaminants still stuck to the antibody from the purification process. If that is the case, then some of your antibodies have CDRs unavailable for protein binding on the chromatin you are studying. That might be why sometimes you get better precipitations than other times. One option to remove any "hitchhiker" antigens stuck to your antibodies is to bind the antibodies to a Protein A column and wash them with a high salt wash (for example, 2 M NaCl) to remove any contaminants.
How many cells are you using? Remember in a typical ChIP you may get very little DNA 5 to 50 ng total starting from 10 million cells. Getting higher concentration may be indicative of failure and high background. You need to test the quality of your ChIP by qPCR on known targets and include negative controls
Last week we got 3 ng using a mixture of monoclonal antibodies against a known TF and we had almost 50 times more in the IgGs negative control. However the qPCR on several known targets were highly enriched compared to input and to the ChIP from IgG. Don’t be misled by the concentration of your ChIPed DNA. The problem is that you need at least 5 to 10 ng for NGS and if you outsource that to a company they may ask you for 50 ng total.
You can Check the ENCODE guidelines to assess the quality of the antibody. Just google ENCODE guidelines.
What you experienced is nothing new for the community doing ChIP frequently. One additional point of caution could be that what you get ultimately comes as the DNA indirectly bound to the bead via a chain of Protein A, the Antibody and the protein to which the DNA was bound to. So take good care of using fresh stock of beads. Older beads tend to have the coat of protein A removed. Hence while the Ab will bind both protein A and the specific protein, it will still be washed away while washing of the beads as it no more sticks to the beads. However, in such case you will also not get much of the DNA in negative controls.
Are you doing X-ChIP or MNase-ChIP? In any case is your chromatin properly sheared and recovered? Check for this point as well.
If you want to know which proteins are binding to your site of interest, you should try to perform PiCH experiment but it's a lot of set up on a single copy target.
http://www.ncbi.nlm.nih.gov/pubmed/19135898
If you try to find this out by ChIP first, no sure you'll get enough proteins, and if you hope to be target specific, sure Pich is better
good luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides