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.

Quantification of interactions among circadian clock proteins via surface plasmon resonance

“…In previous studies, interaction of clock proteins have been investigated with various assays such as co‐immunoprecipitation (Kume et al ., ; Lee et al ., ; Brown et al ., ; Duong et al ., ), luciferase reporter gene assay (Griffin et al ., ), pull‐down assays (Nader et al ., ; Jia et al ., ), yeast two hybrid (Griffin et al ., ), chromatin immunoprecipitation assay (Ye et al ., ), and fluorescence anisotropy (Huang et al ., ). Some of these assays, such as co‐immunoprecipitation, pull‐down, and yeast two hybrid, are complicated and costly, and they do not provide quantitative information (Jia et al ., ). ELISA‐like assays such as protein probing, gel filtration column (Phizicky and Fields, ), and protein domain microarrays (Kaushansky et al ., ) require high amounts of sample and hence are not economical (Chavane et al ., ).…”

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

“…The advantage of analyzing purified protein complexes is the ability to identify specific interacting proteins and posttranslational modifications that may otherwise go undetected in large-scale global analyses. This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6)(7)(8)(9)(10)(11)(12)(13). These experiments have included various types of affinity tags and have been overlaid with RNAi genetic screens (14,15).…”

Deletion of an Enhancer in FGF5 is Associated With Ectopic Expression in Goat Hair Follicles and the Cashmere Growth Phenotype

“…The c-FOS protein is a member of the FOS protein family [59]. The dual-luciferase reporter gene assay is widely used to study promoter activity, transcription factors, intracellular signaling, protein interactions [60], miRNA regulation [61], and target site recognition [62]. The dual reporter gene assay based on re y (Photinus pyralis) and sea kidney (Renilla reniformis, also known as marine pansy) luciferases can improve experimental accuracy by normalizing results and reducing technical differences [63].…”