Chromatin Immunoprecipitation (ChIP)
Chromatin Immunoprecipitation (ChIP) is one of the most widely used techniques used to study epigenetics. The assay determines whether a certain protein-DNA interaction is present at a given location, time and under different conditions. DNA and proteins are constantly moving within cells. In vivo crosslinking using a formaldehyde solution is able to covalently stabilise the protein-DNA complexes which form thereby allowing ChIP to demonstrate an insight into the protein-DNA complexes which exist at a certain time point. An appropriate antibody is required for the immunoprecipitation step. Following the immunoprecipitation of protein-DNA complexes, analysis can be conducted using PCR, qPCR, DNA microarrays or DNA sequencing.Below is a general protocol for ChIP with recommendations.
- Non-biotinylated primary antibody against DNA-bound protein of interest
- Biotinylated secondary antibody
- Lysis buffer
- Dilution buffer
- Wash buffer
- Chelating resin
- 37% formaldehyde solution
- 1M glycineLeupeptin
- Phenylmethylsulfonyl fluoride (PMSF)
- Streptavidin agarose or magnetic beads
- PCR kit
- Dimethyl sulfoxide
- DNA purification kit
- Deionised water
- Optional: MNase
- Crosslink protein-DNA complexes by incubating cells with 37% Formaldehyde diluted to 1% final concentration with shaking for 15 min at room temperature.
- Quench the crosslinking by adding 1M glycine diluted to a final concentration of 125 mM. Shake for 5 min at room temperature.
- Harvest the cells by centrifuging at 4 °C for 5 min, 1,000 x g.
- Wash cell pellet twice with 10 ml of PBS 4 °C.Note: samples can be stored overnight at -70 °C if required.
- Add protease inhibitors to the Lysis buffer (10 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 mM PMSF).6. Lyse the cells by resuspending the pellet in 500 μl of Lysis buffer per 5 x 106. Pipette up and down to resuspend the cells. Incubate on ice for 10 min.Note: from this step onwards, keep samples on ice.
- Shear chromatin to an average fragment size of 200 – 1,000 bp by sonicating samples. Keep chromatin on ice at all times. Optimisation for this step will be required, different cell lines have different sonication requirements.Note: do not pulse for >30 s at one time to ensure proteins are not denatured. Alternatively, chromatin can be fragmented by enzymatic digestion using MNase.
- Transfer 500 μl of each sample to a 1.5 mL microcentrifuge tube.
- Centrifuge lysates for 10 min using a refrigerated centrifuge at 12,000 x g. Collect the supernatant in a clean tube for immunoprecipitation. Discard pellet.
- Use 50 μl of each sample to determine fragment size and DNA concentration.
- Dilute supernatant with 1 ml of Dilution Buffer (containing 10 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 mM PMSF). Add Non-biotinylated primary antibody to the sample. Incubate for 15 min at room temperature in an ultrasonic bath. Add 1 – 10 μg of antibody per 25 μg of DNA.Note: if antigen is expected to have low level expression, incubate overnight at 2 °C - 8°C with rotation.
- Add 5 μg of Secondary antibody and incubate for 15 min at room temp in an ultrasonic bath.
- Add 50 μl of Streptavidin beads (agarose or magnetic) to the samples and rotate for 30 min at 2 °C - 8°C.Note: save approx. 5% of your sample before adding the beads to act as your input control.
- If using agarose collect the beads by centrifugation at 12,000 x g for 1 min.If using magnetic beads collect the beads by leaving the tube in the magnet for 2 minutes.
- Wash 4 times with 1 ml Wash buffers pre-cooled to 2 °C - 8°C each time. Vortex samples to resuspend beads between washes.
- Reverse crosslinking by adding 100 μl of Chelating Resin Solution after the last wash directly to the beads and pipette up and down for 10 s. Boil sample for 10 min in a temperature controlled water bath.
- Microcentrifuge at 12,000 x g for 1 min at room temperature and transfer the supernatant to a clean microcentrifuge tube.
- Add 120 ml of deionised water to beads. Pipette up and down for 10 s, centrifuge for 1 min and collect the supernatant. Pool with supernatant in step 17.Note: samples can be stored at -20 °C - -70 °C if required.
- Concentrate and purify the DNA preparation using a phenol-chloroform extraction or a spin column designed for DNA purification. Resuspend DNA in 50 μl of deionised water.Note: samples can be stored at -20 °C - -70 °C if required.
- Use 2 – 10 μl of DNA sample to analyse using PCR or real-time PCR.