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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: 9927

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Cover of Oncogene, featuring study using the protocol.

Apr 2012

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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.

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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

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