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Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

Jia S, Peng J, Gao B, Chen Z, Zhou Y, Fu Q, Wang H, Zhan L - PLoS ONE (2011)

Bottom Line: The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Transfusion Medicine, 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. 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.

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Design of the DLR-PD assay for the quantitative analysis of PPIs.Schematic diagram of the DLR-PD assay. Quantitative PPIs can be measured after the pull down of biotinylated Fluc-tagged prey proteins using SA-PMPs and bioluminescence detection of the biotinylated Fluc-tagged prey and Rluc-tagged bait proteins bound to the beads, respectively. SA, Streptavidin.
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pone-0026414-g001: Design of the DLR-PD assay for the quantitative analysis of PPIs.Schematic diagram of the DLR-PD assay. Quantitative PPIs can be measured after the pull down of biotinylated Fluc-tagged prey proteins using SA-PMPs and bioluminescence detection of the biotinylated Fluc-tagged prey and Rluc-tagged bait proteins bound to the beads, respectively. SA, Streptavidin.

Mentions: For the easy and efficient analysis of PPIs, we designed a novel dual luciferase reporter pull-down (DLR-PD) assay by combining the biotinylated Fluc pull-down assay with the DLR assay system (Fig. 1). A biotinylated protein pull-down assay, based on specific biotin-streptavidin interactions, is a small-scale affinity purification technique. The binding of biotin to streptavidin is the strongest noncovalent interaction known in nature. The high affinity of biotin for streptavidin allows for the simple, efficient, one-step purification of biotinylated proteins under high-stringency conditions [9].


Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

Jia S, Peng J, Gao B, Chen Z, Zhou Y, Fu Q, Wang H, Zhan L - PLoS ONE (2011)

Design of the DLR-PD assay for the quantitative analysis of PPIs.Schematic diagram of the DLR-PD assay. Quantitative PPIs can be measured after the pull down of biotinylated Fluc-tagged prey proteins using SA-PMPs and bioluminescence detection of the biotinylated Fluc-tagged prey and Rluc-tagged bait proteins bound to the beads, respectively. SA, Streptavidin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198426&req=5

pone-0026414-g001: Design of the DLR-PD assay for the quantitative analysis of PPIs.Schematic diagram of the DLR-PD assay. Quantitative PPIs can be measured after the pull down of biotinylated Fluc-tagged prey proteins using SA-PMPs and bioluminescence detection of the biotinylated Fluc-tagged prey and Rluc-tagged bait proteins bound to the beads, respectively. SA, Streptavidin.
Mentions: For the easy and efficient analysis of PPIs, we designed a novel dual luciferase reporter pull-down (DLR-PD) assay by combining the biotinylated Fluc pull-down assay with the DLR assay system (Fig. 1). A biotinylated protein pull-down assay, based on specific biotin-streptavidin interactions, is a small-scale affinity purification technique. The binding of biotin to streptavidin is the strongest noncovalent interaction known in nature. The high affinity of biotin for streptavidin allows for the simple, efficient, one-step purification of biotinylated proteins under high-stringency conditions [9].

Bottom Line: The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Transfusion Medicine, 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. 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.

Show MeSH