Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation

BioTechniques

Cai Huang¹  Ken Jacobson¹ 

[1] 1Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA;

关键词: protein-protein interactions;    BirA;    streptavidin;    ZZ domain;   

DOI  :  10.2144/000113550

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown