Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag.
Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled efficient and large-scale purification of native protein complexes. The SF-TAP tag, a modified version of the TAP tag, allows a fast and straightforward purification of protein complexes from mammalian cells. It consists of a tandem Strep-tag II and a FLAG epitope (SF-TAP). The SF-TAP tag allows a native elution of protein complexes without proteolytic cleavage needed in the original TAP procedure. Besides the SF-TAP protocol, the principal idea of a pathway mapping by subsequent tagging of copurified proteins is demonstrated for the interactome of the MAPKKK Raf.
Gloeckner
Christian Johannes
CJ
Department of Protein Science, Helmholtz Zentrum München-German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
Boldt
Karsten
K
Schumacher
Annette
A
Ueffing
Marius
M
D000345:Affinity Labels / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000345:Affinity Labels; C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins