Identification of DNA binding proteins by chromatin immnunoprecipitation adapted for yeast cells.
Method Article
ChIP (Chromatin Immunoprecipitation)
https://doi.org/10.1038/protex.2018.143
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posted 29 Jan, 2019
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Identification of DNA binding proteins by chromatin immnunoprecipitation adapted for yeast cells.
REAGENTS
Formaldehyde solution (F8775-500ML, SIGMA ALDRICH)
TBS buffer: 20 mM Tris-HCl pH 8.0, 0.5 M NaCl
Lysis buffer : 50 mM HEPES-KOH pH 7.9, 40 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium desoxicholate, 1 mM PMSF, 1 mM benzamidine and Complete Mini protease inhibitor (Roche Diagnostics)
Dynabeads Protein G (10004D, invitrogen)
HA-probe (F-7) antibody (SC-7392, Santa Cruz Biotechnology Inc.)
Monoclonal GFP antibody (11814460001, Roche Diagnostics).
PBS : 150 mM NaCl, 40 mM Na2PO4, 10 mM NaH2PO4
Tween 20 (P7949-100ML, SIGMA ALDRICH)
Elution buffer: 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS
Proteinase K (03115879001, Roche Diagnostics)
High Pure PCR product purification kit (11732676001, Roche Diagnostics).
SYBR® Premix Ex TaqTM Tli RNase H Plus Green with ROX (RR420A,Takara).
EQUIPMENTS
Genie-2 shaker (Scientific Industries)
BioRuptor Diagenode sonicator
DNA Engine Peltier Thermal Cycler (Bio Rad)
Chromatin Immunoprecipitation assay
Crosslinking and sample collection
Collect approximately 5 × 108 cells from a exponentially growing yeast culture
Crosslink the cells by adding formaldehyde 1% (v/v) to the growth medium and incubate 15 min
Stop crosslinking adding glycine 125 mM and incubate during 5 minutes
Collect the cells by centrifugation at 4000xg and washed four times with 25 mL of cold TBS buffer at 4ºC
Cells can be stored at -20ºC
Inmunoprecipitation
Resuspend the cell pellet in 300 μL of Lysis buffer
Lyse the cells by vortexing with glass beads for 30 min at 4°C with Genie-2 (Scientific Ind.) and supplement with lysis buffer to a final volume of 600 μL
Fragment the chromatin by sonication using a BioRuptor Diagenode with discontinuous pulses during 10 min
Centrifuge the sample at 13400xg for 15 min at 4°C
Keep 20 μL from the supernatant as a control of whole-cell extract (Input)
Incubate the remaining volume with orbital rotation for 2 h at 4°C with Dynabeads Protein G (Invitrogen) previously bound to a specific antibody
Wash the beads 4 times in PBS containing 0.02% (v/v) Tween 20
Elute the bound protein with two times 40 μL of Elution buffer by heating at 65°C for 8 min
Deproteinization, DNA purification and qRT-PCR
Revert the cross-linking by overnight incubation at 65°C shaking
Digest the eluted sample for 90 min at 37°C with 0.33 mg/mL proteinase K (Roche Diagnostics)
Purify the DNA using the High Pure PCR product purification kit (Roche Diagnostics)
Analyse the co-immunoprecipitated DNA in triplicate by quantitative PCR in a DNA Engine Peltier Termal Cycler (Bio Rad) using the SYBR® Premix Ex TaqTM Tli RNase H Plus Green with ROX (Takara)
Binding values indicates the specific enrichment of the analysed promoter region in the immunoprecipitated sample compared to the whole cell extract (Input) using the intergenic region as a control, calculated with the ΔΔCT method. Values are relative to the no tag control strain (value of 1 equivalent to no specific enrichment of the analysed region)
Schmittgen, T.D. and Litvak, K.J. (2008) Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108doi:10.1038/nprot.2008.73
GEN PROMOTER ANALYSED REGION
CLN2 –753 to –428
RNR1 –725 to –442
FKS1 –556 to –304
MNM1 –755 to –456
The authors declare no competing financial interests
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 29 Jan, 2019
You are reading this latest protocol version
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