Chromatin IP
Josh Friedman/Nir Rubins
April 2006
Chromatin Immuno Precipitation (ChIP)
1. Thaw frozen chromatin quickly at 37C, than put on ice.
2. Spin in microfuge, max RPM x 5 min at 4C. Take supernatant leave cellular debris.
Use 8-12 µg chromatin DNA and 2µg antibody per IP (determine empirically).
3. Pre-clear chromatin: add 125 µl of well-mixed protein-A or protein-G agarose to each
chromatin sample (choice of protein-A or –G depends on origin of antibody). Dilute with ChIP
dilution buffer up to 1 ml and rotate 1 hour at 4C.
4. Spin down (all spins 15 sec pulse in microfuge) and use supernatant in IP.
5. Add antibody: 1-4 µg affinity purified or monoclonal antibody and incubate o/n at 4C.
6. Block protein: Wash protein-A/G agarose several times with ChIP dilution buffer before use.
Then add BSA and fish DNA in ChIP dilution buffer and rotate o/n at 4C.
Final amounts:
50µl protein-A/G agarose per IP
BSA to 1 mg/ml
(Roche BSA 20mg/ml)
Fish DNA to 0.1 mg/ml
(Invitrogen Herring sperm DNA 10mg/ml)
Eg: 6 samples:
300µl protein-A/G agarose
50µl of 20mg/ml BSA
10µl of 10mg/ml fish DNA
590µl ChIP dilution buffer
Next Day:
7. Precipitate: add of 50µl of blocked protein-A/G agarose to each chromatin sample and rotate at
4C for 30-60 minutes. Mix well between withdrawals.
8. Wash: 15 sec pulse to pellet agarose, add 1 ml wash buffer, rotate 5 min at RT.
Wash buffers in order: TSE I, TSE II, ChIP buffer III, TE.
9. Elute: add 100 µl freshly-made ChIP elution buffer to final pellet and rotate 10 min at RT.
Spin, keep supernatant. Repeat elution once and combine eluates.
10. Un-crosslink: add 8 µl 5M NaCl per 200 µl eluate (final concentration 192 mM NaCl) and
incubate at 65C for > 4 hours.
11. Add 8 µl 1 M Tris-Hcl, pH 6.5, 4 µl 0.5 M EDTA, and 1 µl 10 mg/ml proteinase K. Incubate
for 1 hour at 45C.
12. Purify un-crossed chromatin using Qiagen PCR purification kit. Elute in 50µl EB.
13. Do Q-PCR and quantify enrichment relative to non-specific genomic sequence. For example,
use primers to the 28S gene as a non-specific sequence, and calculate enrichment from CT’s as
follows: 2^[(28SChIP - YourGeneChIP) - (28SInput - YourGeneInput)].
Chromatin IP
Josh Friedman/Nir Rubins
April 2006
1. ChIP dilution buffer: 0.01%SDS, 1.1% Triton-X 100, 167 mM NaCl, 16.7 mM Tris-HCl, pH
8.1
To make 50 ml:
i. 50 µl 10% SDS
ii. 5.5 ml 10% Triton-X 100
iii. 835 µl 1M Tris-HCl, pH 8.1
iv. 1.67 ml 5M NaCl
2. TSE I: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl
To make 50 ml:
i. 500 µl 10% SDS
ii. 5 ml 10% Triton X-100
iii. 200 µl 0.5M EDTA
iv. 1 ml 1M Tris-HC, pH 8.1
v. 1.5 ml 5M NaCl
3. TSE II: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl
To make 50 ml:
i. 500 µl 10% SDS
ii. 5 ml 10% Triton X-100
iii. 200 µl 0.5M EDTA
iv. 1 ml 1M Tris-HCL, pH 8.1
v. 5 ml 5M NaCl
4. ChIP Buffer III: 0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10 mM Tris-HCl,
pH 8.1
To make 50 ml:
i. 2.5 ml 5M LiCl
ii. 5 ml 10% NP-40
iii. 500 mg deoxycholic acid
iv. 100 µl 0.5M EDTA
v. 500 µl 1M Tris-HCl, pH 8.1
5. Elution buffer: 1% SDS, 0.1 M NHCO3
To make 5 ml:
i. Add 420 mg NaHCO3 to 45 ml water
ii. Take 4.5 ml of this and add 0.5 ml 10% SDS