Stu1-3xFLAG immunoprecipitation from benomyl treatment, Cdc20-AID treatment, and DMSO control
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ABSTRACT: Log-phase Stu1-3xFLAG Cdc20-AID OsTIR1 cells were grown in 3 conditions: 0.1% DMSO (control, asynchronous), 30 ug/mL benomyl with 0.1% DMSO (metaphase arrest), or 1 mM auxin (indole-3-acetic acid) with 0.1% DMSO (metaphase arrest) for 2.5 hours at 23C prior to harvesting. Harvested yeast were washed in water supplemented with 0.2 mM PMSF, pelleted, and resuspended in lysis buffer (Buffer H 0.15): 25 mM HEPES pH 8.0, 150 mM KCl, 2 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.1% NP-40, 15% glycerol, supplemented with protease and phosphatase inhibitors. Yeast pellets were frozen in liquid nitrogen and lysed using a Freezer Mill (SPEX, Metuchen NJ). Lysate was clarified via ultracentrifugation at 24,000 RPM (98,000 x g) for 90 minutes and the protein layer was extracted with a syringe. This extract was incubated with magnetic a-M3DK antibody (targeting Stu1-3xFLAG) conjugated Dynabeads (GenScript) for 3 hours at 4C with rotation, washed in lysis buffer 5 times, washed 2 times in 50 mM Tris pH 8.3, 75 mM KCl, 1 mM EGTA, and eluted with 0.2% RapiGest (Waters Corporation, Milford MA) in 50 mM ammonium bicarbonate for mass spectrometry processing.
INSTRUMENT(S): Orbitrap Fusion ETD
ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)
SUBMITTER:
Susan Biggins
PROVIDER: MSV000099081 | MassIVE |
SECONDARY ACCESSION(S): PXD068187
REPOSITORIES: MassIVE
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