This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
ChIP (Chromatin Immunoprecipitation)
Carmen Bano
Cell Cycle, Universitat València
Corresponding Author
Juan Carlos Igual
Cell Cycle, Universitat València
Corresponding Author
Inma Quilis
Cell Cycle, Universitat València
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Identification of DNA binding proteins by chromatin immnunoprecipitation adapted for yeast cells.
Keywords
Chromatin Immunoprecipitation
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
Version History
CURRENT STATUS: Posted
Version 1
Posted 29 Jan, 2019
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Method Article
ChIP (Chromatin Immunoprecipitation)
Carmen Bano
Cell Cycle, Universitat València
Corresponding Author
Juan Carlos Igual
Cell Cycle, Universitat València
Corresponding Author
Inma Quilis
Cell Cycle, Universitat València
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 29 Jan, 2019
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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