⮝ Full datasets listing
PXD077404
PXD077404 is an original dataset announced via ProteomeXchange.
Dataset Summary
| Title | Kinetochore-microtubule attachments are strengthened by CENP-T stabilization of Stu2 at kinetochores |
| Description | Kinetochore proteins were purified by immunoprecipitation of the Flag tag on the C-terminus of Cnn1 (NM033126_040426_SBY24005A; NM033126_040426_SBY24005B) or Dsn1 (NM033126_040426_SBY8253A; NM033126_040426_SBY8253B) from Saccharomyces cerevisiae lysate from benomyl-arrested mitotic cells expressing Cnn1-6His-3Flag (SBY24005) or Dsn1-6His-3Flag (SBY853). Yeast cells were harvested and resuspended in Buffer H (25mM HEPES, pH 8.0, 150mM KCl, 2mM MgCl2 , 0.1mM EDTA pH 8.0, 0.1% NP-40, 15% glycerol) supplemented with protease and phosphatase inhibitors. Resuspended yeast pellets were pelleted in liquid nitrogen and lysed using Freezer Mill (SPEX, Metuchen, NJ). Lysate was clarified by ultracentrifugation at 98,000 g for 90 minutes, and the protein layer was extracted with a syringe. This extract was incubated with magnetic alpha-M3DK antibody conjugated to Dynabeads (Invitrogen, Waltham, MA) for 3 hours at 4C with rotation. Dynabeads were washed with 10X the bead volume of Buffer H 5 times (the last 2 times omitting DTT). For mass spectrometry, kinetochores were eluted from the Dynabeads by gentle agitation with Flag peptide (1mg/ml) used at 30% bead volume. The kinetochores eluted were precipitated with 20% TCA overnight. The next day, the eluate was spun at 16,000g at 4C for 30 minutes, followed by washing with 1N Acetone-HCl, air-drying at room temperature, resuspending in 10 ul of 1X sample buffer with 5% beta-ME, and boiling for 5 minutes. Each sample was run on a 4-12% Bis-Tris gel (Thermo Fisher Scientific) for 5 minutes at 100V. The gel was stained overnight with 0.1% Coomassie G-250 and destained the following day. The gel section above the 3Flag peptide band was excised for mass spectrometry analysis. |
| HostingRepository | MassIVE |
| AnnounceDate | 2026-07-09 |
| AnnouncementXML | Submission_2026-07-09_12:22:19.858.xml |
| DigitalObjectIdentifier | |
| ReviewLevel | Non peer-reviewed dataset |
| DatasetOrigin | Original dataset |
| RepositorySupport | Supported dataset by repository |
| PrimarySubmitter | Fred Hutch Proteomics Core |
| SpeciesList | scientific name: Saccharomyces cerevisiae W303; NCBI TaxID: 580240; |
| ModificationList | Phospho |
| Instrument | Orbitrap Eclipse |
Dataset History
| Revision | Datetime | Status | ChangeLog Entry |
|---|---|---|---|
| 0 | 2026-04-20 16:38:09 | ID requested | |
| ⏵ 1 | 2026-07-09 12:22:20 | announced |
Publication List
| no publication |
Keyword List
| submitter keyword: Immunoprecipitation, phopshorylation, kinetochore, Saccharomyces cerevisiae, DatasetType:Proteomics |
Contact List
| Susan Biggins | |
|---|---|
| contact affiliation | Fred Hutchinson Cancer Center |
| contact email | sbiggins@fredhutch.org |
| lab head | |
| Fred Hutch Proteomics Core | |
| contact affiliation | Fred Hutchinson Cancer Research Center |
| contact email | pgafken@fredhutch.org |
| dataset submitter | |
Full Dataset Link List
| MassIVE dataset URI |
| Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://massive-ftp.ucsd.edu/v12/MSV000101512/ |
