relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system蛋白质相互作用的相对量化使用双荧光素酶记者拉试验系统.pdfVIP

Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System 1 1 1 2 1 1 3 Shuaizheng Jia , Jianchun Peng , Bo Gao , Zhongbin Chen , Yong Zhou , Qiuxia Fu , Haiping Wang , Linsheng Zhan1* 1 Beijing Institute of Transfusion Medicine, Beijing, China, 2 Beijing Institute of Radiation Medicine, Beijing, China, 3 Department of Blood Transfusion, Hospital 307 of Chinese People’s Liberation Army, Beijing, China 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. Here