The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain.
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.
Dixon
Andrew S
AS
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, 84108, USA.
Lim
Carol S
CS
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D021381:Protein Transport; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein