Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed

GPCRs Interaction Measurement by Fluorescence Resonance Energy Transfer (FRET)

通过荧光共振能量转移（FRET）测定G卵白偶联受体家族（GPCR）的相互作用

Manuel D. Gahete Manuel D. Gahete
RL Raúl M. Luque
JC Justo P. Castaño

发布: 2012年11月20日第2卷第22期 DOI: 10.21769/BioProtoc.293 浏览次数: 9522

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Apr 2012

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用生物正交化学标记制备蛋白裂解物用于点击反应及凝胶内荧光分析

利用生物正交化学标记制备蛋白裂解物用于点击反应及凝胶内荧光分析

Yaxin Xu and Tao Peng

2024年11月20日 1264 阅读

高灵敏且可调控的 ATOM 荧光生物传感器：用于检测细胞中蛋白质靶点的亚细胞定位

高灵敏且可调控的 ATOM 荧光生物传感器：用于检测细胞中蛋白质靶点的亚细胞定位

Harsimranjit Sekhon [...] Stewart N. Loh

2025年03月20日 1406 阅读

Ub-POD：用于识别人细胞中E3泛素连接酶底物的泛素特异性邻近标记技术

Ub-POD：用于识别人细胞中E3泛素连接酶底物的泛素特异性邻近标记技术

Urbi Mukhopadhyay [...] Sagar Bhogaraju

2025年06月20日 1389 阅读

Abstract

This is a protocol to determine the physical interaction of a G-protein coupled receptor (GPCR) with itself (homodimerization) or with other GPCR (heterodimerazation) using fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between the electronic excited states of two dye molecules (in this case, CFP and YFP) in which excitation is transferred from a donor molecule (CFP) to an acceptor (YFP) molecule without emission of a photon that can be used to determine interaction among YFP- and CFP-tagged GPCRs. Nowadays, FRET microscopy technique can be used to determine interaction between any proteins that retain biological function when expressed as a fusion to the fluorescent protein.

Keywords: Fluorescent microscopy (荧光显微镜) search Hormonal receptors (激素受体) search Protein interaction (蛋白质相互作用) search Somatostatin (生长抑素) search High-resolution (高分辨率) search

Materials and Reagents

  1. A cell line lacking the expression of the GPCR of interest
  2. Expression plasmid containing E-CFP
  3. Expression plasmid containing E-YFP
  4. Expression plasmid containing E-YFP and E-GFP coupled in frame (CFP-YFP or Positive control)
  5. Expression plasmid containing the first GPCR of interest tagged with E-CFP (GPCR1-CFP)
  6. Expression plasmid containing the second GPCR of interest tagged with E-YFP (GPCR2-YFP)
  7. Lipofectamine 2000
  8. 4% paraformaldehyde
  9. PBS
  10. Fluoromount
  11. Poly-l-lysine

Equipment

  1. Round coverslips coated with poly-l-lysine
  2. Nikon Eclipse TE2000 E scope equipped with a 400 DCLP dichroic filter (Chroma)
  3. ORCA II BT digital camera

Software

  1. MetaMorph software (Imaging Corporation)
  2. Image J software

Procedure

English
中文翻译

文章信息

版权信息

© 2012 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Gahete, M. D., Luque, R. M. and Castaño, J. P. (2012). GPCRs Interaction Measurement by Fluorescence Resonance Energy Transfer (FRET). Bio-protocol 2(22): e293. DOI: 10.21769/BioProtoc.293.

ris ris Download Citation in RIS Format

分类

分子生物学 > 蛋白质 > 蛋白质-蛋白质相互作用

生物化学 > 蛋白质 > 荧光

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件