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→Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads
==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
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.
* 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.
** LiCl Immune Complex Wash Buffer (Catalog # 20-156), 3-5 washes
** TE Buffer (Catalog # 20-157), two washes
==== Elution of Protein/DNA Complexes ====
===== Prior to starting this section: =====