Chromatin Immunoprecipitation (ChIP) Assay
Materials
1.
1% formaldehyde in PBS
2.
100 µM Tris, pH 9.4, 10 mM DTT
3.
Buffer I
10 mM Hepes, pH 6.5
0.25% Triton X-100
10 mM EDTA, pH 8.0
0.5 mM EGTA, pH 8.0
4.
Buffer II
10 mM
200 mM
10 mM
0.5 mM
5.
6.
7.
8.
9.
Hepes, pH 6.5
NaCl
EDTA, pH 8.0
EGTA, pH 8.0
Lysis Buffer
50 mM
1%
10 mM
1X
Tris, pH 8.1
SDS
EDTA, pH 8.0
protease inhibitors
20 mM
1%
2 mM
150 mM
1X
Tris, pH 8.1
Triton X-100
EDTA, pH 8.0
NaCl
protease inhibitors
20 mM
150 mM
1%
2 mM
0.1%
Tris, pH 8.1
NaCl
Triton X-100
EDTA, pH 8.0
SDS
20 mM
500 mM
1%
2 mM
0.1%
Tris, pH 8.1
NaCl
Triton X-100
EDTA, pH 8.0
SDS
Dilution buffer
TSE I
TSE II
Buffer III
10 mM Tris, pH 8.1
0.25M LiCl
1% NP-40
1% Deoxycholate
1 mM EDTA, pH 8.0
10.
Extraction Buffer
0.1M NaHCO3
1% SDS
Methods
1.
Wash cells 2 times with PBS (RT) and fix with 1% formaldehyde at RT for 10
minutes.
2.
Rinse cells twice with ice-cold PBS and scrape cells into 100 µM Tris-HCl (pH
9.4), 10 mM DTT. (108 cells per ml). Incubate for 15 min at 30°C and spin 5' at 2,000
RPM.
3.
Wash cell pellet with ice-cold PBS, then buffer I then buffer 2. (1ml per 108 cells)
4.
Resuspend cells in -.3 ml lysis buffer.
5.
Sonicate 10 times for 10 secondes each. Centrifuger for 10 min at 14 K and
collect supernatant.
6.
Dilute 1:10 in dilution buffer (this is the soluble chromatin).
7.
Preclear by adding 2 µg of sonicated salmon sperm DNA, 20 µl preimmune
serum and 45 µl 1:1 protein A sepharose 10 1.0 ml of soluble chromatin and then rocking
for 2 hours at 4°. Spin and collect supernatant.
8.
Immunoprecipitate with specific antibody overnight and then add 45 µl of
proteins and 2 µg of salmon sperm DNA. Rock for 1 hour at 4°C.
9.
Wash for 10 minutes each in TSE I, TSE III and buffer III.
10.
Wash three times with TE.
11.
Extract three times with 1% SDS,0.1M NaHCO3. Save and poll the three eluates.
12.
Heat eluates to 65°C for 6 hours to reverse formaldehyde cross-linking and then
purify DNA using Qiaquick column.