162
edits
Changes
→Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads
* It may be possible to reduce the incubation time of the IP. This depends on many factors
(antibody, gene target, cell type, etc.) and will have to be tested empirically.
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
supernatant fraction.
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
