Chromatin Immunoprecipitation (ChIP) Assays

• Wash cells twice with PBS at room temperature, resuspending to approximately 5x10⁵ cells/ml (approximately 2x10⁷ cells total). Add formaldehyde to a final concentration of 1% and incubate at room temperature for 10 minutes.
• Terminate cross-linking reactions by adding glycine to a final concentration of 0.125 M.
• Pellet cells (2,000 RPM, 5 minutes) and wash once with ice cold PBS.
• Resuspend cells in 6 ml Lysis Buffer (sc-45000) by mixing gently.
• Collect crude nuclear extract by microcentrifugation at 2,000 RPM, 5 minutes.
• Wash again with PBS. Pellet may be frozen or processing may be continued as follows:
  1. Resuspend pellet in ~1.9 ml Lysis Buffer High Salt (sc-45001) and transfer to 2 ml microcentrifuge tube for the sonication step.
  2. Sonication conditions should be optimized since results may vary using different sonifiers. The following conditions were established by using a Sonics VC130 with a 3 mm tip probe.
  3. Sonicate on ice at power output setting = 5–6, continuous mode, 4 times at 30 second intervals.
  4. Centrifuge extract for 15 minutes, 10,000 rpm at 4° C and save supernatant (chromatin).
  5. Determine protein concentration of supernatant.
  6. For the IP step we recommend using 100–500 µg supernatant and 0.1-1 µl TransCruz reagent (0.2–2 µg).

NOTE: Investigators may wish to consider using the primary antibody conjugated to sepharose or magnetic beads as an alternative to using secondary immunoprecipitation reagents (e.g., Protein A-Agarose) as described here. Combining primary antibodies directed to different epitopes of the same protein may be advantageous in some cases.

• Preclear the chromatin solution by adding 50 µl Protein A/G PLUS-Agarose (sc-2003) and incubate for 30 minutes at 4° C. Centrifuge at full speed for 5 minutes at 4° C.
• Add primary antibody to the supernatant and incubate overnight at 4° C.
• Add 50 µl Protein A/G PLUS-Agarose (sc-2003) and incubate for 2 hrs at 4° C.
• Harvest beads by centrifugations at 12,000 rpm for 20 seconds and place tube in ice.
• Wash beads twice with 1 ml Lysis Buffer High Salt (sc-45001).
• Wash pellet four times with Wash Buffer (sc-45002).
• Resuspend beads in 400 µl Elution Buffer (sc-45003).
• Reverse cross-links by incubating tube in a 67° C water bath, mixing occasionally over two hours. Remove beads by centrifugation and continue incubating supernatant at 67° C overnight.
• Centrifuge for 3 minutes at 10,000 rpm to remove any residual beads and save supernatant.
• To isolate DNA, extract supernatant once with 500 µl phenol/chloroform/isoamyl alcohol (25:24:1), vortex thoroughly and separate phases by centrifuging tube for 3 minutes at 14,000 rpm.
• Save the aqueous phase, back extract the organic phase once with 100 µl 10 mM Tris, 1 mM EDTA, pH 8.1 (TE) and pool aqueous phases.
• Extract pooled aqueous phase with 600 µl chloroform/isoamyl alcohol.
• DNA may be concentrated by using commercially available kits.