title: An in vivo crosslinking approach to isolate protein complexes from Drosophila embryos.
abstract: Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains--small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.
authors.affiliation: Department of Biological Sciences, Murray State University.
authors.forename: Ming
authors.initials: M
authors.lastname: Gao
authors.affiliation: Department of Biological Sciences, Murray State University.
authors.forename: Patrick
authors.initials: P
authors.lastname: McCluskey
authors.affiliation: Department of Biological Sciences, Murray State University.
authors.forename: Sudan N
authors.initials: SN
authors.lastname: Loganathan
authors.affiliation: Department of Biological Sciences, Murray State University; aarkov@murraystate.edu.
authors.forename: Alexey L
authors.initials: AL
authors.lastname: Arkov
mesh_terms: D000818:Animals; D002498:Centrifugation / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004330:Drosophila / Q000737:chemistry* / Q000196:embryology*; D029721:Drosophila Proteins / Q000302:isolation & purification*; D004625:Embryo, Nonmammalian / Q000737:chemistry; D047468:Immunoprecipitation / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D029721:Drosophila Proteins