Chromatin immunoprecipitation,ChIP
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In a typical Chromatin Immunoprecipitation (ChIP) reaction, cross-linking of target protein and DNA is performed by adding formaldehyde to growing cells, and chromatin is prepared, sheared by sonication, and precleared to reduce nonspecific immunoprecipitation. Immunoprecipitation is performed with a specific antibody. After elution of the protein-DNA complexes from protein A- or protein G-agarose resin, the samples are heated to reverse the covalent cross-links. The DNA fragments are purified and analyzed by PCR or real-time PCR.
Cell lysis buffer:
- • 0.1M Tris-HCl
- • 85mM KCl
- • 0.5% NP-40
- • *1mM PMSF
- • *0.01mg/ml aprotinin
- • *0.01mg/ml leupeptin
Note: Ingredients labeled with * should be added right before each use.
Nuclei lysis buffer:
- • 50mM Tris-HCl
- • 10mM EDTA
- • 1% SDS
- • *1mM PMSF
- • *0.01mg/ml aprotinin
- • *0.01mg/ml leupeptin
Note: Ingredients labeled with * should be added right before each use.
Dialysis buffer:
- • 2 mM EDTA
- • 50 mM Tris-Cl pH=8.0
- • 0.2 % Sarkosyl (for polyclonal antibodies only)
IP washing buffer:
- • 100 mM Tris-Cl pH=9.0 (8.0 for monoclonal antibodies)
- • 500 mM LiCl
- • 1% NP-40
- • 1% deoxycholic acid
Elution buffer
- • 50 mM NaHCO3
- • 1% SDS
DAY 1
1. Add 37% formaldehyde (stock solution) in cell culture medium to a final concentration of 1% and slowly shake the cells on rotating platform for 10 min at RT.
2. Quench cross-linking by adding 100 μl of 1.375 M glycine /ml medium
3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and continue shaking for 5min at RT.
4. Appropriately remove the culture medium. Wash cells twice with pre-chilled PBS and scrape the cells with PBS into 15ml conical tubes. Centrifuge at 2,000 rpm for 2 min at 4°C.
5. Resuspend the pellet with 10 ml cell lysis buffer (cold) and incubate for 10 min on ice.
6. Centrifuge at 5,000 rpm for 5 min, 4°C. Carefully discard the supernatant with pipet.
7. Resuspend the pellet with 1 ml nuclei lysis buffer (chilled) and incubate for 10 min on ice.
8. Proceed with sonication while keeping samples on ice. We use sonication condition of 15s pulse followed by a 1 min rest interval and total sonication period is 5 min. The time and number of pulses will vary depending on sonicator, cell type and extent of crosslinking. DNA fragments should ideally be 300-1000 bp in size.
9. Transfer chromatin to 1.5ml tubes and centrifuge at 14,000 for 10min, 4 °C or freeze at −80°C for a later use.
10. Carefully transfer the supernatant to new tubes.
11. To preclear the chromatin (two ways):
a. Add 50 μl of protein A/G-agarose beads/salmon sperm DNA to the chromatin and rotate for 1 h at 4°C.
b. Add 10-15μl blocked Sapha A cells for every 1X107 cells and incubate on rotating platform for 15 min, 4 °C.
12. Centrifuge the chromatin at 14,000 rpm for 5 min at 4°C. Transfer the supernatants to clean tubes. Note: Aliquot samples for control. (negative and mock)Samples volumes are better to be 200- 500μl.
13. Add specific antibodies to the chromatin samples and rotate overnight at 4°C.
DAY 2
14. If you are using monoclonal antibodies or a polyclonal antibody from a species other than rabbit, add 1μg of specific secondary antibody and incubate on rotating platform for 1h.
15. Corresponding to step 11:
a. Add 50 μl of protein A/G-agarose beads/salmon sperm DNA beads to the chromatin samples on ice. Rotate for 2 h at 4°C.
b. Add 10 μl of blocked Staph A cells to each sample. Incubate on the rotating platform at room temperature for 15 minutes.
16. Centrifuge the samples at 14,000 rpm for 2 min at 4°C.
17. Save 15 μl from the supernatant of IgG sample as "input" for later use.
18. Carefully discard the supernatants of all samples.
19. Wash pellets twice with 1 ml of dialysis buffer and 4 times with IP washing buffer. For each wash, incubate samples on rotating platform for 3min and centrifuge for 1min and carefully discard the supernatant. Efficient washing is critical to reduce background.
20. Elute antibody/protein/DNA complexes by adding 50 μl of IP elution buffer at RT and Vortex for 15 min at setting 3.
21. Centrifuge at 14,000 rpm for 3 min at RT and transfer supernatant to clean tubes.
22. Repeat Steps 20-21 once
25. Centrifuge samples at 14,000 rpm for 5 minutes to remove agarose beads or Staph A cells. Transfer supernatants to clean tubes.
26. Add 1 ul of high concentration RNase A (10 mg/ml) and 5M NaCl to a final concentration of 0.3 M. Incubate samples in the 67°C waterbath for 4-5 hours to reverse formaldehyde crosslinks.
27. Add 2 and a half volumes of ethanol and precipitate at -20°C overnight.
DAY 3
28. Centrifuge samples at 14,000 rpm for 15-20 minutes at 4°C. Remove residual ethanol and allow pellets to air dry completely.
29. Dissolve each pellet in 100 ul of TE buffer, then proceed to PCR assay and agarose gel electrophoresis.