Chromatin Immunoprecipitation (ChIP)

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 immunomagnetic beads 
37% Formaldehyde Solution 1M Glycine Leupeptin 
Aprotinin 
Phenylmethylsulfonyl fluoride (PMSF)
Streptavidin magnetic beads or agarose beads 
PCR kit 
Phosphate-buffered saline (PBS);
Dimethyl sulfoxide (DMSO); 
DNA purification kit 
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: Magnetic separator

Procedure

Crosslink the protein-DNA complexes by incubating the cells with 37% Formaldehyde diluted to 1% final concentration and incubate with cells on a rocking or shaking device for 15 minutes at room temperature. 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).
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). 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.
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. 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.
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.
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.
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.
Microcentrifuge at 12,000 x g for 1 minute at room temperature and transfer the supernatant (~80 μL) to a clean microcentrifuge tube.
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).
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).
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.