ChIP Assay Protocol

Assemble the following components:

•Cells,grown in 10 cm dishes

•Room Temp.PBS

•Cold PBS

•Cold Sonication Buffer (1% SDS,10 mM EDTA,50 mM Tris-HCl pH 7.5)

Prepare sonication buffer as follows: 10 ml of 10% SDS,2 ml of 500 mM EDTA,5 ml of 1 M Tris,pH 7.5,83 ml nuclease-free water

•Protease inhibitors (200mM PMSF in isopropanol,2 mg/ml Aprotinin,2 mg/ml Leupeptin)

•37% reagent grade Formaldehyde

•1.25 M glycine

•1% Agarose gel/EtBr

1.Transfect,induce,or grow 1 to 5x10⁶ cells in 10 cm dish.

2.Rinse cells with PBS 2 times

THE FOLLOWING STEPS WITH FORMALDEHYDE SHOULD BE PERFORMED IN A FUME HOOD.

3.Layer 5 ml of 1% formaldehyde in PBS over cells (make this solution fresh by adding 270μl of 37% formaldehyde to each 10 ml of PBS).

4.Wait 10 min.at room temp.

5.Add 550μl of 1.25 M glycine,swirl gently to mix.

6.Wait 5 min.

7.Aspirate solution and wash with PBS 2 times.

8.Prepare Sonication buffer + protease inhibitors by adding 2μl of PMSF,5μl of Aprotinin,and 10μl of Leupeptin for each 1 ml of Sonication buffer.You will need to prepare 0.5 ml for each plate you are working with.

9.Add 0.5 ml of Sonication buffer + protease inhibitors to each plate,wait 1 min.,and scrape cells with sterile scraper.Pipet into a 2 ml eppendorf tube,and PLACE ON WET ICE for 10 min.

THE FOLLOWING STEPS SHOULD BE PERFORMED ON ICE,AS MUCH AS POSSIBLE.

10.Sonicate at setting of 10,15 times for 10 second pulses each.Place on dry ice,then wet ice for 1-2 min.after each pulse.

11.verify fragmentation of chromatin by running 8μl of the sample on a 1% agarose gel.

12.While gel is running,centrifuge sample for 10-15 min.at top speed in a refrigerated microfuge.

13.Supernatant should be transferred to a fresh microfuge tube.

14.Add another 2μl of PMSF,and either proceed directly to immunoprecipitation or add 100μl of 50% glycerol and quick freeze in liquid N₂ and store at 80℃for future use.