title: Optimized Affinity Capture of Yeast Protein Complexes.
abstract: Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented.
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: John
authors.initials: J
authors.lastname: LaCava
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: Javier
authors.initials: J
authors.lastname: Fernandez-Martinez
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: Zhanna
authors.initials: Z
authors.lastname: Hakhverdyan
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004586:Electrophoresis; D046911:Macromolecular Substances / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*
chemical_list: D000345:Affinity Labels; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins