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Chromatin Immunoprecipitation (ChIP) Protocol

Chromatin Immunoprecipitation (ChIP) assay is an important method for transcriptional regulation monitoring with uncovered knowledge of interactions between specific proteins and a genomic DNA region. Given the fact that DNA-binding proteins (including transcription factors and histones) in living cells can be cross-linked to the DNA that they are binding, ChIP is used to immunoprecipitate the protein–DNA complex out of cellular lysates by an appropriate antibody. The immunoprecipitated complexes are collected by centrifugation, and proteins are eluted for further analysis such as western blot and LC/MS. Nucleic acid analysis is achieved through PCR, RT-PCR, cDNA sequencing and microarray. This protocol provides a detailed procedure to achieve successful ChIP assay in a fast and efficient manner.

Reagents:

  • 37% formaldehyde
  • 1 M glycine
  • Cell lysis buffer: 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, with 1× protease inhibitor cocktail.
  • PBS: pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4.
  • 10% Chelex 100

Equipments:

  • Sonicator
  • Ultrasonic bath
  • Rocking or shaking device
  • Eppendorf tube rotator
  • Refrigerated microcentrifuge
  • Gel electrophoresis apparatus
  • PCR apparatus

Steps:

Cross-link and cell collection

1. Add 27 μL 37% formaldehyde per 1 mL culture medium to obtain a final concentration at 1%, incubate the cells for 10 min at room temperature while slowly shaking on a rocking or shaking device.

2. Quench cross-link by adding 141 μL 1 M glycine per 1 mL culture medium to obtain a final concentration at 125 mM, incubate the cells for 5 min at room temperature.

3. For floating cells: collet cells into a 15-mL conical tube, centrifuge at 2,000 × g at 4°C for 5 min. Discard the supernatant, and wash the cells twice with cold PBS.

Skip to step 5.

4. For adhesive cells: remove the medium, wash the cells twice with cold PBS. Scrape the cells into PBS in a 15-mL conical tube, centrifuge at 2,000 × g at 4°C for 5 min.

Lysis and sonication

5. Discard the supernatant, add 1 mL cold cell lysis buffer per 1 × 107 cells. Resuspend the pellet by pipetting up and down for several times, and incubate on ice for 10 minutes.

6. Sonicate to shear the chromatin into 0.5~1 kb fragment. Avoid foam and keep the sample on ice.

Notes:

  • Sonication conditions should be optimized depend on the sample and sonicator model. Typically, six 15-sec pulses followed by 45-sec rest periods at output 6.0 have been found to work. For analysis the fragment size, extract a small volume of total DNA from an aliquot of sheared chromatin, and then analyze on an agarose gel (1~2%).
  • The volumes is suggested to be ≤ 1 mL to maintain sonication efficiency.

7. Centrifuge at 12,000 × g at 4°C for 10 min, and retain the supernatant.

8. Transfer 0.5 mL supernatant into a fresh 1.5 mL tube for DNA fragment analysis.

Immunoprecipitation

9. Add relevant antibodies or normal IgG to the sample and incubate for 15 min in an ultrasonic water bath at room temperature.

Notes:

  • Choose IgG from the same species where the antibodies were produced.
  • The incubation time should be optimized depend on the antibody and antigen. For antigens with a low level of expression, the incubation could be performed at 4°C overnight on a rotating platform .

10. Prepare Protein A-agarose beads: wash the beads three times with 1 mL lysis buffer (without protease inhibitor), centrifuge at 2000 × g for a few seconds at 4°C and then discard the supernatant.

11. Dilute beads 1:1 with lysis buffer and add 50 μL to the sample.

12. Incubate at 4°C for 30~45 min in a rotating platform.

13. Centrifuge at 2000 × g for 1 min at 4°C and then discard the supernatant.

14. Wash the beads for four times with 1 mL lysis buffer (without protease inhibitor), discard the supernatant.

DNA purification and analysis

15. Resuspend the washed beads with 100 μL 10% Chelex 100.

16. Pipette up and down for several seconds, and then boil the sample for 10 minutes.

17. Centrifuge at 12,000 × g for 1 min at room temperature, and transfer the supernatant into a fresh 1.5 mL tube.

18. Purify DNA using a DNA purification kit or by standard phenol: chloroform extraction protocol.

19. Dissolve DNA with 50 μL ddH2O, and use 2~10 μL of the DNA sample (25% of the reaction volume) in the PCR reactions

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References:

1. Nelson JD, et al., Protocol for the fast chromatin immunoprecipitation (ChIP) method., Nat Protoc., 2006;1(1):179-85.
2. Carey MF, et al., Chromatin immunoprecipitation (ChIP)., Cold Spring Harb Protoc.,2009(9):pdb.prot5279.

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