Chromatin immunoprecipitation (ChIP) protocol from Drosophila embryos
Overview
Target DNA binding protein that has been cross-linked to chromatin (which has been sheared) will be purified using antibodies raised against the specific target protein. Staphylococcus aureus cells (SAC) will be used to purify antibody/chromatin complex via protein A expressed on the bacterial cell surface. Protein A coupled to a support could be used but SAC is cheaper and it has a higher binding capacity than the equivalent amount of protein A beads that are commercially available.
Fixed chromatin solution will be precleared with SAC to remove non-specific binding of components before addition of antibody to the chromatin solution. After sufficient time for antibody/chromatin interaction this complex is purified using a fresh batch of SAC. The antibody/chromatin/SAC complex is washed and the antibody/chromatin complex is eluted using a SDS/low salt buffer. RNAase is added to remove any RNA and the cross linking is reversed by heating. Following an ethanol precipitation, protein is removed using proteinase K treatment and phenol/chloroform extraction. After another ethanol precipitation, a solution containing enriched DNA fragments (which were bound by the target protein) is produced.
This protocol was developed by Rob White's laboratory. This is a modification of the Farnham laboratory mouse protocol (http://genomecenter.ucdavis.edu/farnham/farnham/).
Unless otherwise stated, all centrifugations are at full speed in a standard microfuge.
Equipment and reagents
- Sigma 4K10 bench top centrifuge
- 10ml lyophilised Zysorbin (fixed and killed S.aureus Protein A positive strain) (Invitrogen; Cat. No. 10-1051-1)
- Bovine Serum Albumin (Fraction V), 50 mg/ml stock in water and filter sterilised (Sigma; Cat No. A-7906)
- 50 U/ml Proteinase K, recombinant PCR grade (Roche Diagnostics; Cat. No. 3115887)
- Phenol/Chloroform/Isoamyl alcohol (25:24:1) (BDH; Cat. No. 436732A)
- Glycogen, molecular biology grade (Roche Diagnostics; Cat. No. 901393)
- Pepstatin (Calbiochem; Cat. No. 516482)
- Aprotinin saline solution (Sigma; Cat. No. A6279)
- Leupeptin (Sigma; Cat. No. L2884)
- AEBSF* (Sigma; Cat. No.A8456)
- Nonidet P40 substitute (Sigma; Cat. No. 74385)
*See solutions and reagents
Solutions and reagents
Dialysis buffer
| Reagent stock concentration | Working concentration required | Amount required / 50 ml |
|---|---|---|
| 500mM EDTA.Na2, pH 8 | 2 mM | 200 µl |
| 1M Tris.HCl, pH 8 | 50 mM | 2.5 ml |
| Sarkosyl* | 0.2% | 100 mg |
| Water | Make to 50 ml |
This buffer should be made up the day before as the detergents take a while to dissolve. Heating at 37 °C helps.
*Omit for monoclonal antibodies.
10 x PBS; pH 7.4
- 80 g Sodium chloride
- 2 g Potassium chloride
- 14.4 g Disodium hydrogen orthophosphate
- 2.4 g Sodium dihydrogen orthophosphate
Make to a total volume of 1 litre
PBS/SDS/BME solution
| Reagent stock concentration | Working concentration required | Amount required / 10 ml |
|---|---|---|
| 10X PBS | 1X | 1 ml |
| 10% SDS | 3% | 3 ml |
| beta-mercaptoethanol | 10% | 1 ml |
| Water | Make to 10 ml |
Nuclear lysis buffer
- 50 mM Tris.HCl, pH 8.1
- 10 mM EDTA.Na2
- 1% SDS
- + protease inhibitors
Autoclave without SDS and then add appropriate amount of SDS from 10% stock.
Protease inhibitors:
| Protease inhibitor | Stock concentration | Working concentration | Amount required per ml | Comments |
|---|---|---|---|---|
| Aprotinin | 2.2 mg/ml | 3.3 µg/ml | 1.5 µl | Dissolve in water, store at 4 °C. |
| Leupeptin | 10 mg/ml | 10 µg/ml | 1 µl | Dissolve in water, store at 4 °C. |
| Pepstatin | 2 mg/ml | 4 µg/ml | 2 µl | Dissolve in methanol, store at -20 °C. Heat to 48 °C to dissolve before storage. |
| AEBSF, hydrochloride* | 100 mM | 1 mM | 10 µl | Dissolve 100 mg in 4.18 ml water, store at 4 °C. This is a non-toxic alternative to PMSF |
*4-(-2-Aminoethyl) benzenesulfonlyfluoride, HCl
IP Dilution Buffer
| Reagent stock concentration | Working concentration required | Amount required / 50ml |
|---|---|---|
| 10% SDS | 0.01% | 50 µl |
| 20% Triton X-100 | 1.1% | 2.75 ml |
| 500 mM EDTA.Na2, pH 8 | 1.2 mM | 120 µl |
| 1M Tris.HCl, pH 8 | 16.7 mM | 835 µl |
| 4M NaCl | 167 mM | 2.09 ml |
| Water | Make to 50 ml |
IP wash buffer
| Reagent stock concentration | Working concentrationrequired | Amount required / 50ml |
|---|---|---|
| 1M Tris.HCl, pH 9* | 100 mM | 5 ml |
| 1M LiCl | 500 mM | 25 ml |
| 10% Nonidet P40 (NP40) | 1% | 5 ml |
| Deoxycholic acid | 1% | 0.5 g |
| Water | Make to 50 ml |
This buffer should be made up the day before because the detergents take a while to dissolve. Heating at 37 °C helps.
*pH 8 for monoclonal antibodies
IP Elution buffer
| Reagent stock concentration | Working concentration required | Amount required / 50ml |
|---|---|---|
| 1M NaHCO3 | 50 mM | 2.5 ml |
| 10% SDS | 1% | 5 ml |
| Water | Make to 50 ml |
TE buffer
| Reagent stock concentration | Working concentration required | Amount required / 50ml |
|---|---|---|
| 1M Tris.HCl, pH 8 | 10 mM | 0.5 ml |
| 500mM EDTA.Na2, pH 8 | 1 mM | 100 µl |
| Water | Make to 50 ml |
5X PK Buffer
| Reagent stock concentration | Working concentration required | Amount required / 50ml |
|---|---|---|
| 1M Tris.HCl, pH 7.5 | 50 mM | 2.5 ml |
| 500mM EDTA.Na2, pH 8 | 25 mM | 2.5 ml |
| 10% SDS | 1.25% | 6.25 ml |
| Water | Make to 50 ml |
Protocol
Preparation of Staphylococcus aureus cells (SAC):
- Resuspend Zymed Protein A reagent in 10 ml water.
- Centrifuge at 5000 rpm (4400 x g) for 5 minutes at 4 °C in 15 ml Falcon tubes (or equivalent) in the Sigma 4K10 bench top centrifuge.
- Discard supernatant and resuspend pellet in 10 ml dialysis buffer.
- Repeat steps 2 and 3.
- Repeat step 2, discard supernatant and resuspend in 3ml PBS/SDS/BME solution. Aliquot into 6 microfuge tubes and boil in water bath for 30 minutes.
- Centrifuge for 5 minutes at room temperature.
- Discard supernatant and resuspend in 1 ml Dialysis Buffer.
- Repeat steps 6 and 7.
- Discard supernatant and resuspend pellets in final volume of 4 ml Dialysis Buffer.
- Store as 100 µl aliquots at -20 °C.
Chromatin immunopurification procedure:
Day 1
- Thaw out 100 µl aliquot of SAC and add 20 µl Bovine Serum Albumin to preblock SAC.
- Incubate overnight at 4 °C on a roller (3 hours is fine).
Day 2
- Centrifuge blocked SAC for 1 minute, discard supernant and resuspend in 100 µl of 1X dialysis buffer. Add 1ml of Dialysis Buffer and invert to mix.
- Repeat step 13.
- Centrifuge for 1 minute, discard supernatant and resuspend in 100 µl of Dialysis Buffer. Store on ice until required.
- To preclear chromatin, thaw out chromatin aliquot and add 15 µl blocked SAC to each chromatin aliquot. Rotate on roller at 4 °C for 15 minutes.
- Centrifuge for 1 minute and transfer supernatant to fresh tube.
- Repeat step 17 and transfer supernatant to fresh tube.
- To fresh tube add chromatin (for Heat Shock Factor experiments we used 20-25 µl) to a total volume of 100 µl (use nuclear lysis buffer to make the volume up). Add 200 µl IP Dilution Buffer. Add serum (for Heat Shock Factor (HSF) we used 1 µl of pre-immune or anti-HSF antiserum). Incubate overnight at 4 °C on roller.
- Set up a fresh aliquot of SAC and block overnight, as per steps 11 to 12.
Day 3
- For monoclonal antibodies only, add 1 µg of an appropiate secondary antibody that binds to Protein A and incubate for an additional hour at 4 °C on roller. Only omit this step when working with a polyclonal antibody that binds to Protein A.
- Process overnight blocked SAC, as per steps 13 to 15.
- Add 10 µl SAC (from step 22) to each IP reaction. Rotate for 15 minutes at room temperature.
- Centrifuge for 1 minute, remove supernatant and discard. Pulse spin, then remove remaining residual fluid. Resuspend pellet in 200 µl Dialysis Buffer using pipette tip. Add 1 ml Dialysis Buffer and invert to mix.
- Incubate on roller at room temperature for 3 minutes.
- Repeat steps 24-25 once.
- Centrifuge for 1 minute, remove supernatant and discard. Pulse spin, then remove the remaining residual fluid. Resuspend pellet in 200 µl IP Wash Buffer using pipette tip. Add 1 ml IP Wash Buffer and invert to mix.
- Incubate on roller at room temperature for 3 minutes.
- Repeat steps 27-28 three times.
- Centrifuge for 1 minute, remove supernatant and discard.
- Resuspend pellet in 150 µl IP Elution Buffer and vortex at setting 3 for 15 minutes at room temperature.
- Centrifuge for 1 minute and transfer supernatant to fresh tube.
- Repeat step 31.
- Centrifuge for 1 minute and pool supernatant with that of step 32. Centrifuge for 5 minutes and transfer to fresh tube.
- Add 1 µl RNase (stock 10 mg/ml, in water) and 22.5 µl 4M NaCl (final concentration 0.3M). Incubate samples at 67 °C for 4-5 hours to reverse cross-linking.
- Cool for 1-2 minutes on ice. Add 812 µl ice-cold ethanol, mix by inversion and store at -20 °C overnight.
Day 4
- Centrifuge at 4 °C for 20 minutes and remove supernatant.
- Pulse spin and remove remaining residual fluid.
- Air dry for 1 hour.
- Resuspend pellet (quite large and white) in 100 µl TE.
- Add 25 µl 5X PK buffer and 1.5 µl proteinase K. Incubate at 45 °C for 2 hours.
- Add 175 µl TE to each sample. Add 300 µl phenol/chloroform/isoAmyl alcohol and vortex to mix.
- Centrifuge for 1 minute and carefully remove upper aqueous layer to fresh tube.
- Add 300 µl chloroform and centrifuge for 1 minute. Carefully remove upper aqueous layer to fresh tube.
- Add 37.5 µl 4M NaCl, 10 µg (0.5 µl) glycogen, and 750µl ethanol (ice-cold). Store overnight at -20 °C.
Please note, instead of perfoming steps 42 to 48, it is possible to purify the genomic DNA using a QIAquick PCR purification kit (Qiagen; Cat. No. 28104), as per the manufacturers instructions. Elution is either performed with water, when the material is to be used for a microarray experiments, or else, the Qiagen supplied elution buffer can be used, e.g., when performing a quality control test. This modification to the method increases yield; hence reducing the background signal after hybridisation for both standard and quality control extractions.
Day 5
- Centrifuge at 4 °C for 20 minutes and remove supernatant.
- Pulse spin and remove remaining residual fluid.
- Air dry pellet (small and white) for 1 hour and resuspend pellet in 30 µl water. Store at -20 °C.
Version 1.2. R. Auburn. (10-08-2006)
