Type-3 BRET, an Improved Competition-Based Bioluminescence Resonance Energy Transfer Assay
Type-3 BRET, an Improved Competition-Based Bioluminescence Resonance Energy Transfer Assay
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DOI：
10.1016/j.bpj.2014.04.061
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发表时间：
2014-06-17
影响因子：
3.4
通讯作者：
Davis, Simon J.
中科院分区：
文献类型：
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作者：
Felce, James H.;Knox, Rachel G.;Davis, Simon J.
我们发现，在传统的，基于竞争的生物发光共振能量转移（BRET）膜蛋白化学计量分析，竞争对手的存在下，可以改变标记的蛋白质密度和人为降低能量转移效率。一种充分表征的单体I型膜蛋白CD86和两种G蛋白偶联受体β（2）AR和mCannR2在这些常规测定中均表现为二聚体，在设计用于规避此类伪影的改进的基于竞争的3型BRET测定中表现出单体行为。
We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors beta(2)AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.