Questions about ChIP protocols - (Sep/11/2010 )

Hi guys,


I recently started to do ChIP assays, but there are a few points in the protocol I use that I do not totally understand:

After fixing my cells with 1% formaldehyde to crosslink proteins to DNA and quenching excess formaldehyde with glycine, cells are rinsed with PBS and scraped in PBS containing protease inhibitors. The cell pellet is then recovered after centrifugation at 1500 rpm and lysed in SDS lysis buffer. The cell lysate is then sonicated and centrifuged. Why exactly do we lyse the cells?

After that, the supernatant is precleared with protein A agarose, then incubated with 5 ug immmunoprecipitation antibody and protein A agarose O/N at 4 degrees Celsius. The protein A agarose-chromatin complex is pelleted at 2000 rpm for 5 minutes and washed with a succession of wash buffers containing low salt, high salt, LiCl, and TE. Why are these washes for exactly? Is it to get rid of non-specific binding of proteins and DNA to the beads? And why do we wash with these specific buffers and in that order?

Protein/DNA complexes are then eluted in a SDS and NaHCO3 buffer, and crosslinking reversed.
For that, we add sodium chloride to each sample, incubate O/N at in a 65 degree Celsius water bath. Then we spin the samples, recover the supernatants, and column purify the DNA. Why are the sodium chloride and incubation at 65 degrees for? After spinning, what exactly do we recover in the supernatant? Why not use proteinase K and RNAse instead?

Thank you very much in advance for your answers.

bioche82 on Sat Sep 11 16:13:16 2010 said: