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Chapters
- 00:00Title
- 00:45Introduction
- 01:17Preparation of the Bait and SILAC Control Strain
- 02:44Grinding of Cells
- 04:05Pre-clearing Cell Lysate
- 05:51Eluting From IgG Beads by TEV Protease Cleavage
- 06:45Trichloroacetic Acid (TCA) Precipitation
- 08:11EtOH/ Acetate Precipitation
- 09:00Sample Analysis by Western Blotting and/ or Silver Staining
- 09:30Representative Results/ Outcome
- 10:15Conclusion
Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.
Tags
Protein ComplexesQuantitative ProteomicsS. CerevisiaeLipidsCellular MembranesCompartmentalizationIntracellular Signaling MoleculesNonvesicular RoutesMembrane Contact Sites (MCSs)Endoplasmic Reticulum (ER)Plasma Membrane (PM)Lipid-synthesizing EnzymesLipid TrafficER-PM MCSsYeast ModelEukaryotesScs2pMolecular PartnersGel ElectrophoresisIn-gel DigestionMass Spectrometry
