MassIVE MSV000093775

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Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength

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Description

Cross-linked samples consist of monomeric SPD-5 which were incubated with either kinase dead or constitutively active PLK-1 plus ATP-MgCl2 in a 150mM KCl, 25mM HEPES, pH7.4 buffer for 2 hours at room temperature. Samples were then cross-linked using 8mM DMTMM for 45min and quenched with 50 mM ammonium bicarbonate for 15 min at RT shaking at 300rpm. Samples ran on a 16-20% SDS-page gel revealing a monomer band and a dimer band in both conditions using a Commassie based stain. Monomer bands were excised, then digested overnight with trypsin (Pierce), reduced with DTT and alkylated with iodoacetamide (Sigma-Aldrich). Samples were cleaned using solid-phase extraction with an Oasis HLB plate, then injected into an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples 1122415-1122420: de-phosphorylated Samples 1123298-1123300: phosphorylated [doi:10.25345/C5BK17125] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Centrosomes ; Cell Division ; Microtubules ; Tensile Forces ; C. elegans

Contact

Principal Investigators:
(in alphabetical order) 		Jeffrey Woodruff, UT Southwestern Medical Center, USA
Submitting User: alemoff
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