title: Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.
abstract: The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.
authors: Beijing Institute of Transfusion Medicine, Beijing, China.
authors: Shuaizheng
authors: S
authors: Jia
authors: Jianchun
authors: J
authors: Peng
authors: Bo
authors: B
authors: Gao
authors: Zhongbin
authors: Z
authors: Chen
authors: Yong
authors: Y
authors: Zhou
authors: Qiuxia
authors: Q
authors: Fu
authors: Haiping
authors: H
authors: Wang
authors: Linsheng
authors: L
authors: Zhan
mesh_terms: D000818:Animals; D002460:Cell Line; D019281:Dimerization; D005240:Feasibility Studies; D048888:Fireflies; D017930:Genes, Reporter; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D008156:Luciferases / Q000235:genetics*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
chemical_list: D050838:Interferon Regulatory Factor-3; D011506:Proteins; D008156:Luciferases