Hello,

The first thing to keep in mind is the quality of the antibodies you use. You should previously check the specificity either by WB or by IF (this is called "primary validation") you should ideally get a single band in the WB, or the band which corresponds with the predicted molecular weight should account for 50% or more of the total lane density including background. Regarding IF, you should get a signal that corresponds to the expected localization. The secondary validation for the antibody, at least in the case of transcription factors, consists in using siRNA to knockdown the protein and then verify that you lost the WB or IF signal. Both of these approaches are used to confirm antibody specificity.

On the other hand, when you do the chromatin immunoprecipitation you should ideally check the enrichment of your protein at known binding sites by real time PCR, to confirm the efficiency of the IP and to be shure you will have enough material for constructing the library for sequencing.

Those are two mayor concerns regarding ChIP-seq.

I´ll leave you a link of a very good articlle regarding these and other considerations you have to have before starting a ChIP-seq experiment:

http://genome.cshlp.org/content/22/9/1813.full.pdf+html

Good luck!

Thank you Franco.

Do you do the sequencing yourself, or do you have a lab, that does this for you? If the second is true, ask them, if they can recommend any protocol (they usually can do that).

Thanks, Christian.

Check out these links:

http://epigenie.com/getting-your-fix-optimizing-chromatin-fixation-for-chip-analysis/

http://epigenie.com/a-beginners-guide-to-chip/

Thank you!