Do you know the amount of chromatin needed per IP for ChIP? Abcam said 25ug of DNA per IP, but I want to try to use much less than that. Do you have suggestions?
Its pretty much relative, although 25-50ug is just enough. Relative; because the expression level of your target gene in your ChIP experimenting cells and primary antibody quality and its efficient interaction with target protein, these two factors influence your final ChIP results.
The real problem is the efficiency of your immunoprecipitation and the sensitivity of your assay. Standard protocols calls for 25ug of DNA (or 25ug of Bradford extimated protein) for reacting with a certain amount of antibody. But then you should obtain an amount of IP DNA sufficient for few or several (depending from your efficiency) real-time PCR amplifications. If your material is limiting and you use a sensitive end-point (as real-time PCR) and you expect a good IP efficiency you can probably reduce the input DNA or protein and (proportionally) the amount of antibody. In any case the best way to proceed is always to perform pilot experiments on putatively positive DNA targets (if available) and/or to test the presence of your target protein in the IP by western blots. For the second kind of pilot experiments you probably need to perform a ChIP on an ABCAM suggested scale.
It is hard to answer this question, because it depends on the downstream application the ChiPped DNA is needed for (target-gene approach? HiSeq?), and how much material you need for that. When possible, performing a 3-log standard curve (1, 10, 100 or 5, 50, 500) and comparing the final yield and quality might help you.
It depends on many things.
First as already mentioned dependes on the downstream application you with to perform. It also depends on the kind of chip you are doing, for example if you are doing histone modification chip or a TF chip. I have succesfully used 2 ugr of plant DNA for histone modification chip followed by deep sequencing (hi-seq) with an abundant histone mark. For TF I have used around 10 ugr .
It is also important to consider that adding more DNA means adding more other substantces to the chip and may inhibit the antibody, so more DNA is not allways better.
If you have a possitive control for your chip you could allways perform a titration of the amount of DNA and determine wich is the most convinient.
As per some of the valuable suggestions above i agree it is hard to answer that what minimum amount of DNA that required per IP. One needs to standardize the protocol according to their need in the lab. If your antibody is really sensitive for the protein then less amounts can also work in some cases.
Good luck
There is no perfect answer to this question - as stated by others, you have to try out what is the best for your protein you IP and the DNA you test for. For one IP I use in general 400 microg (1 microg/microL in Bradford, thus I determine protein concentration) lysate as starting material and solubilize the final DNA pellet in 100 microL. Usually, 0.5 to 1 microL of this is enough for a decent signal in standard PCR.
I wish you good luck for your experiments.
It is better to base your experiment on cell number or other ways to standardize your input - then do a titration. For native ChIP of histone isoforms a few 10 000 cells are enough. We have described the procedure in Cosseau,C. and Grunau,C. (2011) Native chromatin immunoprecipitation. Methods Mol. Biol. 791, 195-212.
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