METHOD FOR ENHANCING AND PROLONGING THE BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET) SIGNAL IN A BRET ASSAY AND A SUBSTRATE SOLUTION FOR USE IN A BRET ASSAY

摘要

An improved BRET assay, wherein the BRET signal is enhanced and/or prolonged. The improved BRET assay comprises the steps of i) adding a substrate to a cell comprising GPCR-Rluc fusion protein and a beta-arrestin-GFP fusion protein, wherein the (beta-arrestin is mutated, ii) adding a ligand to obtain, if possible, a GPCR-Rluc/(beta-arrestin-GFP complex, and iii) measuring a BRET signal to obtain a BRET ratio, wherein the improvement leads to an increased BRET ratio compared with the ratios obtained by use of the same process employing a beta-arrestin-GFP fusion protein wherein the beta-arrestin is the wild type beta-arrestin, or employing a 13-arrestin-GFP fusion protein, wherein the (beta-arrestin is a beta-arrestin specifically mutated so that it acts on the receptor independent of the receptors phosphorylation state. The invention further relates to a stable substrate solution for use in an improved BRET assay.