Chromatin Immunoprecipitation of tagged kinetochore proteins from yeast whole
cell extracts
1. For each sample grow 25 - 50 ml yeast cells to aproximately OD600 0.5 - 1.0.
Usually dilute overnight cultures to .3 in the morning to have cells ready to harvest in
the early afternoon.
2. Crosslink cells with 1% formaldehyde for 15 min at RT with occasional inversion.
(Add 0.67 ml 37% formaldehyde to 25 ml cells - do this in the hood)
3. Quench crosslinking by adding 2.5 M glycine to a final conc. of 125 mM and
incubate for 5 min. (add 1.25 ml to 25 ml culture)
4. Pellet cells and wash once with ~20 ml ice-cold TBS (20 mM Tris HCl, pH 7.6, 150
mM NaCl.). After you dump off TBS, transfer pellet (resuspended in whatever
volume TBS was left) to screw cap centrifuge tube. Spin briefly to pellet and remove
remaining TBS. (Can stop at this point by freezing cell pellets at -80°C).
5. Resuspend pellet in 500 uL ice-cold lysis buffer:
50 mM HEPES/KOH pH7.5
140 mM NaCl
1 mM EDTA
.1% Na - Deoxycholate
1mM PMSF (from 100mM-17 mg/ml stock made fresh in ethanol)
1x LPC
6. Add an equal volume (500 µL) glass beads and lyse by beadbeating 2 x 30 sec, with a
1 min rest between beatings. (in cold room)
7. In the cold room, puncture bottom of tube with a needle and spin lysates into new
tubes (snap top are ok now). Spin lysates for 5 minutes at top speed in
microcentrifuge. Keep everything on ice from now on. Remove supernatant and
resuspend pellet in 500 µL lysis buffer.
8. Shear chromatin by sonicating 5 times for 10 seconds each on setting 3 of the
sonicator - use small probe. Do the samples in order so that they rest in between:
1,2,3,4,...1,2,3,4,...1,2,3,4...
9. Pellet cell debris at 14K for 10 min and decant supernatant to fresh tube. (Can stop at
this point by freezing extracts at -80°C.)
10. Remove one 50 µL aliquot for crude lysate control for ChIP - to determine the equal
presence of all DNA fragments prior to the ChIP, remove one 25 µl aliquot for the
western control, to determine the equal presence of protein prior to ChIP. The aliquot
used for PCR needs to be taken through the post IP reversal of crosslinking - add 200
µL TE/1%SDS and set aside. Add 25 µL 1X sample buffer to sample for westerns
and freeze at -80°C.
11. Prepare magnetic beads for IP: Add 10µL protein G linked Dynabeads to 500µL
lysis buffer. Vortex and put the tubes in magnet holder, then remove buffer. Add
crude lysates to the beads, and add 8µL rabbit anti-myc antibody. Vortex.
12. Incubate 2 hrs on hematology mixer in the cold room.
13. Place tubes in magnet holder and remove supernatant.
14. Wash beads (add wash buffer as indicated below, vortex, quick spin in picofuge, put
tubes back in magnet holder, remove buffer):
2x with 1 ml lysis buffer
1x with 1 ml lysis buffer/500 mM NaCl
1x with 1 ml Chip wash buffer:
10mM Tris/HCl pH8
0.25 M LiCl
0.5% NP-40
0.5% Na-Deoxycholate
1mM EDTA
1x with 1 ml of TE
15. Elute precipitate from antibody beads by adding 100 µL of 50 mM Tris/HCl pH 8.0,
10mM EDTA, 1% SDS and incubating at 65°C for 15 min.
16. Transfer SN from beads to a fresh tube and wash beads with 150 µL TE/0.67% SDS.
Combine this with first eluate. Take a 50 µL aliquot for post IP western control and
add 50 µL 1X protein sample buffer, freeze at -80°C. Incubate the remaining IP
samples, as well as the crude lysate samples, at 65°C for at least 6 hours - I usually do
it overnight.
17. After crosslinks are reversed add 250 µL TE, 5 µg glycogen (10 µL of .5 mg/ml), 100
µg Proteinase K (5 µL of 20 mg/ml) and incubate 2 hr at 37°C.
18. Add 55 µL 4M LiCl and phenol/chloroform extract. Add 1 ml of EtOH and
precipitate DNA at room temerature for 15 min, wash with 75% EtOH, let pellets dry
at RT and resuspend in 50 µL TE + 10 µg RNAaseA. Incubate 2 hr at 37°C.
19. use 1 µL CL DNA and 3 µL DNA in CHIP PCR reactions. (CHIP program in
thermocycler):
95° - 5', 55° - 30", 72° - 1' (95° - 30", 55° - 30", 72° - 1') X 27, 72° - 7'
If you use surestart enzyme use the ARCSS program. Only difference is that the
initial 95C incubation is for 10'. You need this to activate the surestart enzyme.
20. For western controls, incubate samples 30 min at 95°, then load 15uL on a 10%
polyacrylamide gel.