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protocol
Chromatin Immunoprecipitation to Measure Transcription Factor–DNA Interactions
Sponsored,vendor-submitted protocol    Sponsored by R&D Systems    Published in November 2009 (p.7) DOI: 10.2144/000113011

Abstract

Chromatin proteins play crucial roles in DNA repair, replication, and transcription. Chromatin immunoprecipitation (ChIP) has become a powerful technique in gene expression studies, used to assess whether a certain protein-DNA interaction is present at a given location, condition, and time point. R&D Systems ExactaChIP™ Kits are designed to provide a fast, simple method for the identification of genomic DNA target sequences bound by a specific transcription factor.

Chromatin proteins play crucial roles in DNA repair, replication, and transcription. Chromatin immunoprecipitation (ChIP) has become a powerful technique in gene expression studies, used to assess whether a certain protein-DNA interaction is present at a given location, condition, and time point. R&D Systems ExactaChIP™ Kits are designed to provide a fast, simple method for the identification of genomic DNA target sequences bound by a specific transcription factor.

Materials

ExactaChIPKits (R&D Systems)

Specific Assay and Control Antibodies (sufficient for processing 20 samples)

Specific Control Primers Lysis, Dilution, and Wash Buffers Chelating Resin Solution MagCellect Streptavidin Ferrofluid (R&D Systems, Cat# MAG999)


Figure 1. Outline of protocol. (Click to enlarge)



Figure 2. Detection of Nanog binding to the Nanog promoter using the ExactaChIP assay. (Click to enlarge)


Methods

  1. Fix the cells by incubating them with 1% formaldehyde for 15 min at room temperature.

  2. Add 125 mM glycine and swirl for 5 min at room temperature, pellet the cells, and remove the media.

  3. Resuspend the pellet in Lysis Buffer containing protease inhibitors and incubate on ice for 10 min. Note: keep samples on ice from this step forward.

  4. Sonicate the samples to shear chromatin and transfer to 1.5 mL tubes.

  5. Centrifuge the lysates for 10 min at maximum speed. Collect the supernatant in a clean tube.

  6. Dilute the supernatant in 1 mL of Dilution Buffer and add 5 µg of the biotinylated antibodies to the samples. Incubate for 15 min in an ultrasonic bath.

  7. Add Streptavidin beads (magnetic or agarose) and incubate for 30 min at 2–8°C on a rotating device.

  8. Collect the beads and wash them with 1 mL of each of the four Wash Buffers provided. After the last wash, add 100 µL of Chelating Resin Solution and boil for 10 min.

  9. Centrifuge for 1 min at maximum speed and transfer the supernatant to a clean tube.

  10. Add deionized or distilled water to the beads, mix and centrifuge for 1 min at maximum speed, collect the new supernatant, and pool it with the supernatant from Step 9.

  11. Clean up the DNA preparation using a DNA purification kit.

  12. Use 2–25 µL of the DNA samples in standard PCR reactions.

Correspondence

Address correspondence to R&D Systems, Inc. Tel.: (800) 343-7475, [email protected]; R&D Systems Europe, Ltd., +44 (0)1235 529449, [email protected]; R&D Systems China Co., Ltd., (21) 52380373, [email protected]

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