Tandem affinity purification of functional TAP-tagged proteins from human cells.
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.
Gregan
Juraj
J
Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 1, 1030 Vienna, Austria. juraj.gregan@univie.ac.at
Riedel
Christian G
CG
Petronczki
Mark
M
Cipak
Lubos
L
Rumpf
Cornelia
C
Poser
Ina
I
Buchholz
Frank
F
Mechtler
Karl
K
Nasmyth
Kim
K
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
D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; C496644:SGO1 protein, human