| • |
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:
| a) |
Resuspend pellet in ~1.9 ml Lysis Buffer High Salt (sc-45001) and transfer to 2 ml microcentrifuge tube for the sonication step. |
| b) |
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. |
| c) |
Sonicate on ice at power output setting = 5-6, continuous mode, 4 times at 30 second intervals. |
| d) |
Centrifuge extract for 15 minutes, 10,000 rpm at 4° C and save supernatant (chromatin). |
| e) |
Determine protein concentration of supernatant. |
| f) |
For the IP step we recommend using 100-500 μg protein and 0.1-1 μl TransCruz reagent (0.2-2 μg). |
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| 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 PLUSAgarose (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 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. |
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Chromatin Immunoprecipitation (ChIP) Assays |
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