http://ygac.med.yale.edu/
http://ygac.med.yale.edu/mtn/Mammalian_ChIp_protocol.htm
chromatin immunoprecipitation
Chromatin Immunoprecipitation from Mammalian Cell Extracts
1. Crosslink protein DNA complexes in vivo
Grow cells in suspension and collect 5 x 108 cells by low speed centrifugation.
Resuspend cells in 50ml media. In fume hood, add 1.4ml 37% fomaldehyde solution to a
50ml conical tube. Fill tube with cell culture to just below the 50ml line. Incubate at
room temperature for 15min. with occasional inversion. (The extent of cross-linking is
critical and depends on the protein of interest. Too much cross-linking may mask
epitopes and too little cross-linking may lead to incomplete fixation. The concentration
of formaldehyde, the length of cross-linking or the temperature of cross-linking can all be
adjusted.)
2. Quench cross-links
Add 3.4ml 2M glycine to fixed culture and incubate at room temperature for
5min. with occasional inversion.
3. Harvest cells
Centrifuge cells (5min. at 3000rpm) and discard supernatant. Wash cells with
10ml ice-cold 1X TBS and spin cells down, again and discard supernatant. Place cells on
ice.
(Cells can keep on ice for a few hours, if you are collecting many samples for a time
course. Alternatively, cells may be frozen in liquid nitrogen and placed at -80°C).
4. Lyse cells
Resuspend cell pellet gently with a pipette in 10ml RIPA buffer with protease
inhibitors and incubate on ice for 30min. Further disrupt cells by passing them through a
21 guage needle. Add 100l 10mg/ml PMSF and incubate on ice for another 30min.
Transfer lysate to 2ml microcentrifuge tubes (1ml lysate/tube).
5. Shear chromatin
Using a Branson 350 Sonifier with a microtip at a power setting of 7 and a 60%
duty cycle, sonicate extracts for nine 10sec pulses. In between 10sec. pulses, let samples
sit on ice for atleast 2min. This should shear chromatin to a final average size of 500bp.
(Your sonicator will have to be calibrated to yield the desired final average length of
DNA).
6. Clarify samples
Centrifuge samples at max speed for 5min at 4°C. Transfer supernatant to a fresh
1.5ml microcentrifuge tube and centifuge samples again for 15min at max speed at 4°C.
7. Preclear extracts
Add 30l bed volume of Protein A sepharose beads to each tube and incubate on
a rotation wheel for 50min. at 4°C. Centifuge samples at 7500rpm for 2min and then
transfer supernatant to a fresh tube.
8. Immunoprecipitation
Add the primary antibody against the protein of interest to the extract.
(Preliminary immunoprecipitation experiments should be performed to determine the
appropriate amount of antibody to be used). Incubate on ice for 3hrs, then add 30l bed
volume Protein A sepharose beads. Incubate on rotating wheel for 1hr at 4°C.
Centrifuge sample for 2min at 7500rpm at 4°C. Keep 50l of sample for sizing DNA and
add 200l 1%SDS/1X TE to it, discard the rest of the supernatant.
9. Wash immunoprecipitates
Add 1ml RIPA buffer to the beads and incubate for 5min. on rotating wheel at
4°C and then centrifuge at 7500rpm for 2min. Discard supernatant.
Add 1ml RIPA-500 to the beads and repeat incubation and centrifugation.
Add 1ml LiCl/detergent solution to the beads and repeat incubation and
centrifugation.
Add 1ml 1XTBS to the beads and repeat incubation and centrifugation.
10. Elute immunoprecipitates
Add 100l 1%SDS/1X TE, mix and incubate at 65°C for 10min. Centifuge
briefly and transfer eluate to fresh tube and wash beads with 150l 0.67%SDS/1X TE.
Briefly centrifuge and add wash to eluate.
11. Reverse cross-links
Incubate the immunoprecipitates and the total extract material for at least 6hrs at
65°C.
12. Proteinase K treatment
Add 250l Proteinase K solution and incubate for 2hrs at 37°C.
13. Purify DNA
Add 55l 4M LiCl andd 500l 25:24:1 phenol/chloroform/isoamyl alcohol.
Vortex vigorously for 1min. Separate phases by centrifugation at max speed for 10min.
at room temperature. Transfer aqueous phase to a fresh tube and add 1ml 100% ethanol.
Mix and cetrifuge at max speed for 15min. at room temperature. Discard the supernatant
and dry pellet. Resuspend DNA in 10l 1XTE and store at -20°C
Analyze data by PCR assay or microarray analysis.
Solutions:
1X TBS
150mM NaCl
20mM Tris-HCl, pH7.6
RIPA Buffer
10mM Tris-HCL, pH8
140mM NaCl
0.025%NaN3
1% Triton X-100
0.1% SDS
1% Deoxycholic acid
Lysis Buffer-500
10mM Tris-HCL, pH8
500mM NaCl
0.025%NaN3
1% Triton X-100
0.1% SDS
1% Deoxycholic acid
LiCl/detergent wash
0.5% Deoxycholic acid
1mM EDTA
250mM LiCl
0.5% NP-50
10mM Tris-HCl, pH8
Proteinase K solution
1l 20l/l glycogen
5l 20g/l Proteinase K
244.5l 1X TE, pH7.6