Changes

* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, place the entire volume for the number of desired immunoprecipitations in one large tube that will be able to accommodate a volume of 1.1 mL for each IP.

* Each 100 μL will contain ~1 x 106 cell equivalents of chromatin.

18. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.

* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Dilution Buffer containing Protease Inhibitor Cocktail II for the correct number of immunoprecipitations.

19. Add 60 μL of Protein G Agarose for each IP.

* The Protein G Agarose is a 50% slurry. Gently mix by inversion before pipetting.

* This step serves to “preclear” the chromatin, i.e., to remove proteins or DNA that may bind nonspecifically to the Protein G agarose.

* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Protein G Agarose for the correct number of immunoprecipitations.

20. Incubate for 1 hour at 4°C with rotation.

* If different chromatin preparations are being carried together through this protocol, remove

1% of the chromatin as Input from each.

23. Collect the remaining supernatant and dispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet.

24. Add the immunoprecipitating antibody to the supernatant fraction:

* For the positive control, anti-RNA Polymerase, add 1.0 μg of antibody per tube.

* For the negative control, Normal Mouse IgG, add 1.0 μg of antibody per tube.

* For user-provided antibody and controls, add between 1-10 μg of antibody per tube. The appropriate amount of antibody needs to be determined empirically.

25. Incubate overnight at 4°C with rotation.

* It may be possible to reduce the incubation time of the IP. This depends on many factors