Chromatin Immunoprecipitation from Yeast Whole Cell Extracts
互联网
1.Crosslink protein DNA complexes in vivo
Grow 50ml yeast culture to OD600 0.5-1.0.In fume hood,add 1.4ml 37% fomaldehyde solution to a 50ml conical tube.Fill tube with 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.Resuspend cells in 1ml ice-cold 1X TBS and transfer to a 2ml microcentrifuge tube.Centrifuge cells at max speed for 1min.,then 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℃).
4.Lyse cells
Resuspend cell pellet in 400 ml ice-cold lysis Buffer with protease inhibitors.Add an equal volume of cubic zirconium beads and vortex for 10min at 4℃.Let samples sit on ice for 15min.,then move the extract to a fresh 2ml microcentrifuge tube (2ml tube allows for max bead movement while vortexing).Wash the beads with 400 ml ice-cold lysis buffer and vortex for 1min.Add the wash to the lysate.
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℃.Transfer supernatant to a fresh 1.5ml microcentrifuge tube and centifuge samples again for 15min at max speed at 4℃.
7.Preclear extracts
Add 30 ml bed volume of Protein A sepharose beads to extracts and incubate on a rotation wheel for 50min.at 4℃.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 30ml bed volume Protein A sepharose beads.Incubate on rotating wheel for 1hr at4℃ Centrifuge sample for 2min at 7500rpm at 4℃.Keep 50 ml of sample for sizing DNA and add 200 ml 1%SDS/1X TE to it,discard the rest of the supernatant.
9.Wash immunoprecipitates
Add 1ml lysis buffer to the beads and incubate for 5min.on rotating wheel at 4℃ and then centrifuge at 7500rpm for 2min.Discard supernatant.
Add 1ml lysis buffer-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 ml 1%SDS/1X TE,mix and incubate at 65℃ for 10min.Centifuge briefly and transfer eluate to fresh tube and wash beads with 150 ml 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℃.
12.Proteinase K treatment
Add 250 ml Proteinase K solution and incubate for 2hrs at 37℃.
13.Purify DNA
Add 55 ml 4mliCl andd 500 ml 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 ml 1XTE and store at -20℃
Analyze data by PCR assay or microarray analysis.
Solutions:
1X TBS
150mM NaCl
20mM Tris-HCl,pH7.6
Lysis Buffer
0.1% Deoxycholic acid
1mM EDTA
50mM HEPES/KOH,pH7.5
140mM NaCl
1% Triton X-100
Lysis Buffer-500
0.1% Deoxycholic acid
1mM EDTA
50mM HEPES/KOH,pH7.5
500mM NaCl
1% Triton X-100
LiCl/detergent wash
0.5% Deoxycholic acid
1mM EDTA
250mmliCl
0.5% NP-50
10mM Tris-HCl,pH8
Proteinase K solution
1 ml 20 ml/ ml glycogen
5 ml 20 m g/ ml Proteinase K
244.5 ml 1X TE,pH7.6
