Chromatin immunoprecipitation - ChIP

Overview

Chromatin immunoprecipitation (ChIP) is a method used to assess the association between protein and specific DNA regions. ChIP involves the cross-linking of DNA with proteins, fragmentation, and prep of chromatin to then be followed by immunoprecipitation with an antibody to recognize target proteins.

Required materials

• Cross-linking agent

• Protease inhibitors

• Lysis buffer

• Sonicator

• ChIP-suitable Antibodies

• Protein beads

• Wash buffers

• Elution buffer

• Reverse cross-linking buffer

• PCR reagents

• Ultrasonic bath

• Distilled water

Protocol

1. Cross-linking the protein-DNA involves incubating the cells with diluted Formaldehyde on a rocking/shaking device at room temperature.

2. The formaldehyde should then be quenched with diluted glycine.

3. Add protease inhibitor to the lysis buffer, resuspend the cells, and incubate on ice, be sure to keep the samples cold.

4. Prepare the ultrasonic bath for sonication of the samples to shear the chromatin to an average length, optimization may be required.

5. Transfer samples into microcentrifuge tubes and centrifuge using a refrigerated ultracentrifuge.

6. Collect the supernatant in a clean tube.

7. Dilute the supernatant with diluting buffer and add normal IgG to the samples and incubate at room temperature in the ultrasonic bath.

8. Add the secondary antibody and incubate at room temperature.

9. Add streptavidin beads either magnetic or agarose to the samples.

10. Collect and wash the beads with distilled water, collect new supernatant, and pool with supernatant from step 6.

11. Concentrate the DNA preparation with a DNA purification kit.

Results

• To analyze the purified DNA there are a couple of options including ChIP-PCR, ChIP-qPCR, ChIP-seq, and ChIP-chip.

• ChIP-PCR and ChIP-qPCR excel at single-gene analysis which can be used for amplifying and quantifying specific fragments very fast and cost-effectively.

• ChIP-chip analysis utilizes DNA microarray chips to create a genome-wide, high-resolution map of the protein binds and modification.

• ChIP-seq analysis uses NGS technology to align purified DNA with previously annotated whole genomes to identify genome-wide protein binding profiles.