title: Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation.
abstract: Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells.
authors.affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA. cai-huang@uky.edu
authors.forename: Cai
authors.initials: C
authors.lastname: Huang
authors.forename: Ken
authors.initials: K
authors.lastname: Jacobson
mesh_terms: D000818:Animals; D019921:Biotinylation / Q000379:methods*; D016466:CHO Cells; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D011232:Chemical Precipitation; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D005978:Glutathione