SnAvi--a new tandem tag for high-affinity protein-complex purification.
Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.
Schäffer
Ursula
U
Bioinformatics and Molecular Genetics, ZBMZ, Institute for Biology, ZBSA-Center for Systems Biology, Albert-Ludwigs-Universitaet Freiburg, 79104 Freiburg, Germany.
Schlosser
Andreas
A
Müller
Kristian M
KM
Schäfer
Angelika
A
Katava
Nenad
N
Baumeister
Ralf
R
Schulze
Ekkehard
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D029742:Caenorhabditis elegans Proteins / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism; D050987:Vesicle-Associated Membrane Protein 1 / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D029742:Caenorhabditis elegans Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C475157:UBC-25 protein, C elegans; D014452:Ubiquitins; D050987:Vesicle-Associated Membrane Protein 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; C487100:chn-1 protein, C elegans