The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain

• Published in: BioTechniques Vol. 49; no. 1; pp. 519 - 524
• Main Authors: Dixon, Andrew S, Lim, Carol S
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis Group 01-07-2010
• Subjects: Animals; Bcr; Cell Line; Cell Nucleus - metabolism; Chlorocebus aethiops; Cloning, Molecular; COS Cells; DsRed; EGFP; Gene Expression; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; nuclear translocation assay; Protein Interaction Mapping - methods; protein switch; Protein Transport; protein-protein interactions; Proteins - analysis; Proteins - genetics; Proteins - metabolism; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Red Fluorescent Protein
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/000113452

Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein.