162
edits
Changes
→Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads
* 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).
* 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
(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.
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'''** [[High Salt Immune Complex Wash Buffer]] (Catalog # 20-155), '''one wash'''** [[LiCl Immune Complex Wash Buffer]] (Catalog # 20-156), '''3-5 washes'''** [[TE Buffer]] (Catalog # 20-157), '''two washes ''' ''Note: for TE washes use a pipette to carefully aspirate, the beads seem to come off of the magnet easily with this wash''
=== Elution of Protein/DNA Complexes ===