title: Tandem affinity purification of functional TAP-tagged proteins from human cells.
abstract: Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.
authors.affiliation: Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 1, 1030 Vienna, Austria. juraj.gregan@univie.ac.at
authors.forename: Juraj
authors.initials: J
authors.lastname: Gregan
authors.forename: Christian G
authors.initials: CG
authors.lastname: Riedel
authors.forename: Mark
authors.initials: M
authors.lastname: Petronczki
authors.forename: Lubos
authors.initials: L
authors.lastname: Cipak
authors.forename: Cornelia
authors.initials: C
authors.lastname: Rumpf
authors.forename: Ina
authors.initials: I
authors.lastname: Poser
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Kim
authors.initials: K
authors.lastname: Nasmyth
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D034622:RNA Interference
chemical_list: D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; C496644:SGO1 protein, human