Detection of TAP-tagged proteins in Western blot, confocal laser scanning microscopy and FACS using the ZZ-domain.
Tandem affinity purification of protein complexes has become an important tool in the field of proteomic research. Analysis of the proper intracellular localization of TAP-tagged proteins by immunohistochemistry by specific antibodies is often impossible due to the simultaneous detection of the endogenously synthesized native protein. Here we show that the highly specific interaction of the ZZ-domain of Protein A, which constitutes part of the original TAP-tag, to rabbit IgGs can be used to detect TAP-tagged proteins in fixated cells by Confocal Laser Scanning Microscopy just by the use of labeled secondary antibodies. In addition, such interactions can be exploited for the analysis of transfected cells in FACS and Western blot experiments. Thus, we present valuable tools for the analysis of recombinant proteins on the basis of IgG-ZZ interactions, which can be used even if target specific first antibodies are not available or lack sufficient specificity.
Weser
Stephan
S
Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, 40001 Düsseldorf, Germany. stephan.weser@uni-duesseldorf.de <stephan.weser@uni-duesseldorf.de>
Gerlach
Martin
M
Kwak
Daniel Min-Seok
DM
Czerwinska
Malgorzata
M
Gödecke
Axel
A
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D009842:Oligopeptides / Q000032:analysis*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D007074:Immunoglobulin G; D009842:Oligopeptides; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C080396:trypsinogen activation peptide