We identified a protein which modifies H3K9 and seems to form a complex with it, DNA and some other proteins modifying the gene expression of the gene it is bound to. We would like to know which is the binding partner which determines the DNA site where the complex binds to and which other proteins are necessary as part of the complex to make it functional.
I have seen some publications with DNA pool down and Mass Spect, but is there any other option with less disadvantages?
Fundamentally, one has to design some kind of ChIP comparison between cells expressing the protein and not expressing the protein to identify functional binding sites.
Do you know whether the protein binds DNA directly or not?
It is necessary to crosslink the protein complex while it is bound to DNA. Therefore a few questions arise:
1) Do you have an antibody to the protein is native configuration?
2) Does the protein, when bound to H3K9, obscure the usual site to which H3K9 (Chip) antibodies bind?
Identification of the other partners in a complex is difficult when the amounts of material are limiting – hence the use of MS in many cases. If one has a list of suspects, one can use a variety of Co-IP and Western blotting. This can be helpful even when performing MS or MS-MS.
Easiest method is EMSA-2D gel electrophoresis followed by ms sequencing to identify other protein partners! To identify DNA bound site, gradually trim down the DNA during EMSA.
Perhaps ChIA-PET might be helpful. Check it here: http://genomebiology.com/2010/11/2/R22
Some experiments could be chosen
If you want to identify aonther protein in DNA binding complex. Do Re-chip, which uses second specific AB to do another CHIP with first chromatin precipitation. Then use that precipitation to do Western and reverse the crosslink to detect DNA. Sometimes you need do double crosslink at the beginning.
If only to determine the DNA binding site, do chip -chip or chip-seq.
If you have got the binding DNA site sequence, do yeast one hybrid to identify other binding proteins or partners.
If to investigate the function of DNA binding sequence, use Luciferase reporter vector.
I think, that the attached publication coud give some answers.
ChIP seq would be the best way allowing to precipitate your protein linked to DNA before sequencing the DNA bound to your protein of interest.
That are great answers, thanks a lot
@ Gerard: 1) Yes we have an AB, but it is not ChIP grade and we have not tried it for ChIP so far, but it works for Elisa and Western
2) We do not know how it binds, just that it does
As a follow-up: You can test if the Ab will have a chance of succeeding at ChIP, by determining if you can IP the protein from solution with the Ab. Isolate nuclei, use a mild method of dissociating and solubilizing protein, e.g., low concentration of non-ionic detergent, and perform an IP followed by Western. If the Ab can pull down the non-denatured protein from solution, one can hope that the binding site (mAb) or binding sites (polyclonal) are exposed when complexed. If it does not IP, there is little chance ofsuccess at ChIP (or even EMSA).
If you have a specific gene locus to be analyzed, one option is to use our insertional chromatin immunoprecipitation (iChIP) to pull down the locus followed by mass spec (iChIP-MS). You can use zin-finger nuclease (ZFN) or TALEN to make knocking-ins. I hope the link and attachment are helpful, and feel free to contact me if you are interested.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0026109
Have you tested with foot printing to see if really the protein binds DNA directly?
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