A combined approach for the localization and tandem affinity purification of protein complexes from metazoans.
An understanding of a protein's function is greatly aided by the knowledge of its localization in vivo and identification of any interacting partners. Here, we describe a method, based on expression of a genetically encoded fusion protein, that allows for protein localization and affinity purification (LAP) in metazoans. This method makes it possible to rapidly identify transformants and to conduct live imaging of protein localization and dynamics. In addition, the same tag can be used in a modified tandem affinity purification (TAP) procedure to isolate native protein complexes of high purity. The efficacy of this purification procedure allows for the characterization of rare protein complexes that are largely inaccessible to classical biochemical purifications. The LAP strategy should be widely applicable to the characterization of protein function in cellular and developmental contexts in various metazoans.
Cheeseman
Iain M
IM
Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA. icheeseman@ucsd.edu <icheeseman@ucsd.edu>
Desai
Arshad
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000166:cytology; D029742:Caenorhabditis elegans Proteins / Q000302:isolation & purification*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose / Q000379:methods; D049452:Green Fluorescent Proteins / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
D029742:Caenorhabditis elegans Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins