Analytical biochemistry
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Analytical biochemistry · Feb 2009
Comparative StudyDirect comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage.
Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET(1) with Renilla luciferase (RLuc) and coelenterazine h (CLZ, lambda(em) approximately 475 nm) and BRET(2) with the substrate coelenterazine 400a (CLZ400A substrate, lambda(em)=395 nm) as the respective donors. For BRET(1) the acceptor is yellow fluorescent protein (YFP) (lambda(em) approximately 535 nm), a mutant of green fluorescent protein (GFP), and for BRET(2) it is GFP(2) (lambda(em) approximately 515 nm). ⋯ The BRET(2) assay for thrombin was 2.9 times more sensitive compared with the BRET(1) version. Calculated detection limits (blank signal+3sigma(b), where sigma(b)=standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET(1) and 15 pM (0.0005 U) thrombin with BRET(2). The results presented here suggest that BRET(2) is a more suitable system than BRET(1) for studying protein-protein interactions and as a potential sensor for monitoring protease activity.