title: Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.
abstract: It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada.
authors.forename: Rhiannon
authors.initials: R
authors.lastname: Campden
authors.forename: Darlaine
authors.initials: D
authors.lastname: Pétrin
authors.forename: Mélanie
authors.initials: M
authors.lastname: Robitaille
authors.forename: Nicolas
authors.initials: N
authors.lastname: Audet
authors.forename: Sarah
authors.initials: S
authors.lastname: Gora
authors.forename: Stéphane
authors.initials: S
authors.lastname: Angers
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
mesh_terms: D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002352:Carrier Proteins; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits