From Nelson et al, Nature 2006 "Protocol for the fast chromatin immunoprecipitation (ChIP) method
- prepare aliquots of 0.2 million cells/IP, (~200ul in my case)
- Add Ab to pre-cleared chromatin samples, incubate in ultrasonic water bath for 15 mins (must be determined for each Ab used) at 4.c (in cold room).
- Do mock IP, without Ab, or pre-immune serum
- Spin down chromatin, 10 mins @ 12,000g
- Wash 3x protein A beads (20 ul per IP sample) in IP buffer (see paper)
- A wash is in 1ml IP buffer, spin centrifuge 2,000g for a few seconds.
- Dilute beads 1:1 in IP buffer, aliquot 40ul slurry to a clean eppendorf (make sure to add exactly the same amount each time)
- Now take the top 90% of your pre-cleared chromatin and add it to the beads you just prepared , avoid taking the very bottom of the chromatin, to keep sample clean.
- add 100ul 10% wt/vol Chelax 100, slurry direct to the beads