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Method Article
Co-Immunoprecipitation assay for HA-tagged proteins from yeast
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
Co-Immunoprecipitation sassy for tagged proteins using yeast cells
Keywords
Co-Immunoprecipitation HA-tagged proteins yeast
-Lysis buffer: 50 mM Tris-HCl pH 8, 250 mM NaCl, 5 mM EDTA, 0,1% (v/v) Triton X-100, and Complete Mini protease inhibitor (Roche Diagnostics)
-Dynabeads Protein G (10004D, Invitrogen)
- HA-probe (F-7) antibody (SC-7392, Santa Cruz Biotechnology Inc.)
- PBS : 150 mM NaCl, 40 mM Na2PO4, 10 mM NaH2PO4
- Tween 20 (P7949-100ML, SIGMA ALDRICH)
- 2x SDS-PAGE sample solvent: 4% SDS, 20% glycerol, 0.02% bromophenol blue, 0.1 M DTT, 0.125 M Tris-HCl pH 7.5
Sample collection and protein extract
Collect approximately 5 × 108 cells expressing either untagged or HA-epitope tagged versions of protein to immunoprecipitate from exponentially growing yeast cultures.
Collect the cells by centrifugation at 4000xg and wash them once with H2O.
Resuspend the cell pellet in 100 μL of Lysis buffer.
Lyse the cells by vigorous shaking with glass beads for 3 min at 4°C.
Clarify the sample at 13400xg for 5 min at 4ºC.
Keep 10 μl from the supernatant as a control of whole-cell extract (Input).
Dynabeads Protein G preparation
Wash twice 50µL of Dynabeads with PBS containing Tween 0.02%.
Incubate for 30 min at room temperature the dried beads with 10µL of HA-probe (F-7) antibody (Santa Cruz Biotechnology Inc) supplemented with 190µL PBS-Tween 0,02%.
Wash the unbound antibody twice with PBS-Tween 0,02%.
Immunoprecipitation
Incubate the remaining volume of the whole cell extract with Dynabeads-antibody with orbital rotation for 20 min at room temperature.
Keep 10 μL from the supernatant as a control of unbound protein.
Wash the beads 5 times with PBS-Tween 0,02%.
- Elute the immunoprecipitated proteins by boiling the beads for 5 min in 2x SDS-PAGE sample solvent and analyze by SDS-polyacrylamide gel electrophoresis followed by Western blot analysis.
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CURRENT STATUS: Posted
Version 1
Posted 29 Jan, 2019
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Method Article
Co-Immunoprecipitation assay for HA-tagged proteins from yeast
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
Co-Immunoprecipitation sassy for tagged proteins using yeast cells
-Lysis buffer: 50 mM Tris-HCl pH 8, 250 mM NaCl, 5 mM EDTA, 0,1% (v/v) Triton X-100, and Complete Mini protease inhibitor (Roche Diagnostics)
-Dynabeads Protein G (10004D, Invitrogen)
- HA-probe (F-7) antibody (SC-7392, Santa Cruz Biotechnology Inc.)
- PBS : 150 mM NaCl, 40 mM Na2PO4, 10 mM NaH2PO4
- Tween 20 (P7949-100ML, SIGMA ALDRICH)
- 2x SDS-PAGE sample solvent: 4% SDS, 20% glycerol, 0.02% bromophenol blue, 0.1 M DTT, 0.125 M Tris-HCl pH 7.5
Sample collection and protein extract
Collect approximately 5 × 108 cells expressing either untagged or HA-epitope tagged versions of protein to immunoprecipitate from exponentially growing yeast cultures.
Collect the cells by centrifugation at 4000xg and wash them once with H2O.
Resuspend the cell pellet in 100 μL of Lysis buffer.
Lyse the cells by vigorous shaking with glass beads for 3 min at 4°C.
Clarify the sample at 13400xg for 5 min at 4ºC.
Keep 10 μl from the supernatant as a control of whole-cell extract (Input).
Dynabeads Protein G preparation
Wash twice 50µL of Dynabeads with PBS containing Tween 0.02%.
Incubate for 30 min at room temperature the dried beads with 10µL of HA-probe (F-7) antibody (Santa Cruz Biotechnology Inc) supplemented with 190µL PBS-Tween 0,02%.
Wash the unbound antibody twice with PBS-Tween 0,02%.
Immunoprecipitation
Incubate the remaining volume of the whole cell extract with Dynabeads-antibody with orbital rotation for 20 min at room temperature.
Keep 10 μL from the supernatant as a control of unbound protein.
Wash the beads 5 times with PBS-Tween 0,02%.
- Elute the immunoprecipitated proteins by boiling the beads for 5 min in 2x SDS-PAGE sample solvent and analyze by SDS-polyacrylamide gel electrophoresis followed by Western blot analysis.