Changes

===Immunoprecipitation===(taken from Millipore EZ ChIP protocol)

# 15. Prepare enough Dilution Buffer containing protease inhibitors for the number of desired immunoprecipitations and store on ice.## 16. Each IP requires the addition of 900 μL of Dilution Buffer and 4.5 μL of Protease Inhibitor Cocktail II.## 17. Immunoprecipitations should include the positive control (Anti-RNA Polymerase II), and the negative control, (Normal Mouse IgG), and the antibody of interest (user supplied). It is recommended that the user include a negative control IgG of the same species as the antibody of interest.# Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 5) for the number of desired immunoprecipitations and put on ice. If chromatin has been previously frozen, thaw on ice.## * 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.# 21. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).## * Do not spin Protein G Agarose beads at high speeds. Applying excessive g-force may crush or deform the beads and cause them to pellet inconsistently.# 22. Remove 10 μL (1%) of the supernatant as Input and save at 4°C until Section D, step 1.## * If different chromatin preparations are being carried together through this protocol, remove

# 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

# 26. Add 60 μL of Protein G Agarose to each IP and incubate for 1 hour at 4°C with rotation.## * This serves to collect the antibody/antigen/DNA complex.# 27. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove the

# 28. Wash the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of the cold buffers in the order listed below and incubating for 3-5 minutes on a rotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:** Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one wash** High Salt Immune Complex Wash Buffer (Catalog # 20-155), one wash** LiCl Immune Complex Wash Buffer (Catalog # 20-156), one wash3-5 washes** TE Buffer (Catalog # 20-157), two washes