Technical Information

Technical Information

Chromatin Immunoprecipitation (ChIP) Protocol

Introduction to ChIP

Chromatin is composed of proteins, DNA, and RNA. Found in the nucleus of eukaryotic cells, it mediates several central biological processes, such as regulating cell-specific or tissue-specific gene expression and DNA replication and repair. The ChIP assay has become one of the most practical and useful techniques to study the mechanisms of gene expression, histone modification, and transcription regulation. In addition, ChIP assays are particularly useful for the identification of transcription factors and their target genes. This assay determines whether a certain protein-DNA interaction is present at a given location, condition, and time point. The use of an appropriate antibody for the immunoprecipitation step is considered the most critical factor for a successful ChIP assay.1, 2, 3, 4, 5, 6

General Description of this ChIP Protocol

This protocol is intended to provide general guidelines, experimental settings, and conditions for ChIP, the immunoprecipitation of protein-DNA complexes that might be later analyzed by PCR, qPCR, DNA microarrays, or direct DNA sequencing. Specific optimization might be required if instruments differ from those described here. In addition, different cell or tissue samples many express differing protein levels that can require further optimization of the procedure.

The ChIP protocol described here can be conducted in about 2.5 hours, when processing up to 10 different samples. It avoids the use of time-consuming crosslinking-reversal, overnight antibody incubation, and antibody capture with protein A/G agarose that is traditionally used in conventional protocols.7, 8, 9, 10, 11, 12 Antibody incubation is performed in an ultrasonic bath. For antibody capture, the biotin-streptavidin system is employed. This protocol is similar to other published fast/sensitive ChIP protocols.13, 14 For DNA sample clean-up, this protocol includes the use of a chelating resin solution and silica-based columns. These steps greatly reduce the time required to run the experiment. Additional details and applications can be found in a recent report authored by R&D Systems researchers, Medeiros et al. (2009).15

Reagents Required

  • Biotinylated Antibody or non-Biotinylated Antibody plus anti-IgG-biotin (Click for all antibodies suitable for ChIP)
  • Biotinylated Normal IgG (negative control)
  • Lysis Buffer, Dilution Buffer, Wash Buffer, and Chelating Resin (ExactaChIP™ Chromatin IP Buffer Panel, R&D Systems, Catalog # ECP001)
  • 37% Formaldehyde Solution (Sigma, Catalog # 252549; or equivalent)
  • 1M Glycine (Sigma, Catalog # G6388; or equivalent)
  • Leupeptin (Tocris, Catalog # 1167; or equivalent)
  • Aprotinin (Sigma, Catalog # A4529; or equivalent)
  • Phenylmethylsulfonyl fluoride (PMSF; Sigma, Catalog # P7626; or equivalent)
  • Streptavidin magnetic beads or agarose beads (Sigma, Catalog # 85881; or equivalent)
  • PCR kit (Applied Biosystems, Catalog # 4311806; or equivalent)
  • Phosphate-buffered saline (PBS; Life Technologies, Catalog # 10010-023; or equivalent)
  • Dimethyl sulfoxide (DMSO; Tocris, Catalog # 3176; or equivalent)
  • DNA purification kit (Qiagen, Catalog # 28144; or equivalent)
  • Deionized or distilled water
  • Optional: primers for a known target gene (to be used as a positive control if PCR or qPCR is the technique chosen for read-out)

Note: ExactaChIP Chromatin Immunoprecipitation Kits include primary antibody, control antibody, lysis buffer, dilution buffer, wash buffer, and chelating resin solution.

Materials

  • Pipettes and pipette tips
  • 1.5 mL microcentrifuge tubes
  • 15 mL Falcon tubes
  • Sonicator
  • Ultrasonic bath
  • Rocking or shaking device
  • Eppendorf tube rotator
  • Benchtop ultracentrifuge
  • Refrigerated ultracentrifuge
  • Benchtop centrifuge
  • Water bath and heatblock
  • PCR thermocycler
  • If using magnetic beads, the following is also required: Magnet (R&D Systems, Catalog # MAG999; or equivalent)

Precautions

Formaldehyde is flammable, highly toxic, and potentially carcinogenic. Refer to the MSDS from the supplier for handling and hazard information.

The Lysis Buffer should be kept at room temperature.

The Dilution Buffer and Wash Buffers should be kept at 2 °C to 8 °C. It is important that the Dilution Buffer and Wash Buffers are kept cold prior to use.

Procedure

  1. Crosslink the protein-DNA complexes by incubating the cells with 37% Formaldehyde diluted to a 1% final concentration and incubate with cells on a rocking or shaking device for 15 minutes at room temperature.
  2. Quench the Formaldehyde by adding 1M Glycine diluted to a final concentration of 125 mM. Rock for 5 minutes at room temperature, pellet the cells, and remove the media (at this point, samples can be stored overnight at < -70 °C).
  3. Add protease inhibitors to the Lysis Buffer (10 μg/mL Leupeptin, 10 μg/mL Aprotinin, and 1 mM PMSF). Resuspend the cell pellet in 500 μL of Lysis Buffer per 5 x 106 cells. Pipette up and down to resuspend the cells, and incubate on ice for 10 minutes (keep samples on ice from this step forward).
  4. Sonicate the samples to shear chromatin to an average length of about 1 kb (optimization may be required; see the Technical Hints section for details). Transfer 500 μL of each sample to a 1.5 mL microcentrifuge tube.
  5. Centrifuge the lysates for 10 minutes using a refrigerated ultracentrifuge at 12,000 x g. Collect the supernatant in a clean tube and discard the pellet.
  6. Dilute the supernatant by adding 1 mL of Dilution Buffer (containing the same amount of protease inhibitors as in step 3) and add 5 μg of the Antibody or Normal IgG to the samples. Incubate at room temperature for 15 minutes in an ultrasonic bath (alternatively, incubate overnight at 2 °C to 8 °C on a rotating device, if the antigen is expected to have a low level of expression). Add 5 μg of the secondary antibody (for example donkey anti-goat IgG-biotin), incubate at room temperature for 15 minutes in an ultrasonic bath (alternatively, incubate for 1-2 hours at 2 °C to 8 °C on a rotating device). The secondary antibody step is unnecessary when using a biotinylated primary antibody.
  7. Add 50 μL of Streptavidin beads (magnetic or agarose) to the samples and rotate for 30 minutes at 2 °C to 8 °C on a rotating device.
  8. If using magnetic beads, collect the beads by leaving the tube in the magnet for 2 minutes. If using agarose beads, collect the beads by centrifugation at 12,000 x g for 1 minute. Perform 4 washes with Wash Buffers pre-chilled to 2 °C to 8 °C. For each wash, add 1 mL of Wash Buffer. Start with Wash Buffer 1 and finish with Wash Buffer 4. Pipette the beads up and down between each wash.
  9. After the last wash, add 100 μL of Chelating Resin Solution directly to the beads and pipette up and down for about 10 seconds. Boil the sample for 10 minutes using a heatblock or a temperature-controlled water bath.
  10. Microcentrifuge at 12,000 x g for 1 minute at room temperature and transfer the supernatant (~80 μL) to a clean microcentrifuge tube.
  11. Add 120 μL of deionized or distilled water to the beads. Pipette up and down for 10 seconds, centrifuge for 1 minute, collect the new supernatant, and pool it with the supernatant from Step 10 (at this point, samples can be stored at < -20 °C or < -70 °C).
  12. Clean up and concentrate the DNA preparation using a DNA purification kit. Resuspend the DNA in 50 μL of deionized or distilled water. This step increases the yield of PCR fragments by concentrating the DNA in a smaller volume and minimize impurities that might affect the PCR reaction (at this point, samples can also be stored at < -20 °C or < -70° C).
  13. Use 2-10 μL of the DNA sample in the PCR reactions (see the Technical Hints section for details).

Technical Hints

Fixation

The duration of the incubation and the final concentration of the Formaldehyde can affect the efficiency of the procedure. A shorter incubation (e.g. 5 minutes) may improve shearing efficiency, however, it may affect the yield of precipitated DNA.

Experimental Conditions

Stimulation time and conditions should be determined and optimized by the investigator according to the cell type of interest.

Sonicator Settings to Obtain 1 Kilobase Fragments

The sonicator settings need to be optimized by the investigator. For example, DNA of 1 kilobase (kb) average size can be obtained by setting a Heat Systems-Ultrasonics sonicator to 4% output power, 70% duty, output control 3, performing 4 rounds of 15 pulses (2 second pulses), resting the samples on ice water for 2 minutes between rounds, and keeping the samples on ice water at all times. Foaming should be avoided as it might decrease the shearing efficiency. Foaming can be caused by inappropriate position of the sonicator probe (too close to the surface or touching the bottom of the tube).

To determine the DNA fragment average size, run an aliquot of sheared chromatin (after Step 5 of the Procedure) along with a DNA marker on a 1-2% (weight/volume) agarose gel. Stain the gel with Ethidium Bromide and visualize the DNA under UV light.

Antibody Incubation

When using a biotinylated antibody the incubation time can be changed to overnight at 2 °C to 8 °C to increase the efficiency of the immunoprecipitation, if needed. For example, this can be done in case of a limiting number of cells, or for an antigen expressed at low relative levels.

Controls

As a positive control for PCR, DNA prepared from samples prior to immunoprecipitation (whole cell lysates) can be used as total or input DNA.

For additional controls, use an irrelevant antibody, primers designed to anneal to promoters known not to be regulated by the protein of interest, or primers designed to anneal to regions of the gene known not to bind to the protein of interest.

References

  1. Solomon, M.J. & A. Varshavsky (1985) Proc. Natl. Acad. Sci. USA 82:6470.
  2. Bernstein, B.E. et al. (2005) Cell 120:169.
  3. Cosma, M.P. et al. (1999) Cell 97:299.
  4. Fesenfeld, G. & M. Groudine (2003) Nature 421:448.
  5. Dundr, M. et al. (2002) Science 298:1623.
  6. Li, C.C. et al. (2006) Nat. Immunol. 7:692.
  7. Nandiwada, S.L. et al. (2006) J. Immunol. 177:401.
  8. Nelson, J.D. et al. (2006) Nucleic Acids Res. 34:e2.
  9. Becker, P.B. et al. (1999) Chromatin Protocols (Methods in Molecular Biology Vol. 119), Humana Press, Totowa, NJ.
  10. Kuo, M.H. & C.D. Allis (1999) Methods 19:425.
  11. Johnson, K.D.& E.H. Bresnick (2002) Methods 26:27.
  12. Roh, T-Y. et al. (2004) Nat. Biotechnol. 22:1013.
  13. Nelson, J.D. et al. (2006) Nat. Protoc. 1:179.
  14. Dahl, J.A. & P. Collas (2008) Nat. Protoc. 3:1032.
  15. Medeiros, R.B. et al. (2009) BMC Biotechnol. 9:59.

Publications using R&D Systems ExactaChIP Chromatin Immunoprecipitation Kits

  1. Guadall, A. et al. (2011) J. Biol. Chem. 286:7093.
    • Human/Mouse HIF-1 alpha ExactaChIP Chromatin IP Kit (Catalog # ECP1935)

    Pubmed ID: 21193390

  2. Kurita, S. et al. (2010) Oncogene 29:4848.
    • Human GLI-1 ExactaChIP Chromatin IP Kit (Catalog # ECP3324)
    • Human GLI-2 ExactaChIP Chromatin IP Kit (Catalog # ECP3526)
    • Human GLI-3 ExactaChIP Chromatin IP Kit (Catalog # ECP3690)
    • Human/Mouse BID Antigen Affinity-purified Polyclonal Antibody (Catalog # AF860)

    Pubmed ID: 20562908

  3. Yan, W. et al. (2010) J. Biol. Chem. 285:14042.
    • ExactaChIP Chromatin IP Buffer Panel (Catalog # ECP001)

    Pubmed ID: 20189993

  4. Lan, L. et al. (2010) Int. J. Cancer 126:53.
    • Human Snail ExactaChIP Chromatin IP Kit (Catalog # ECP3639)

    Pubmed ID: 19521958

  5. Martorell, L. et al. (2009) Mol. Cell. Biol. 29:5828.
    • Human/Mouse HIF-1 alpha ExactaChIP Chromatin IP Kit (Catalog # ECP1935)
    • Recombinant Human VEGF165 (Catalog # 293-VE)
    • Human VEGF165 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-293-NA)
    • Human NGFI-B gamma/NOR-1/NR4A3 MAb (Clone H7833) (Catalog # PP-H7833-00)

    Pubmed ID: 19720740