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FRET (Fluorescence Resonance Energy Transfer) quantitative detection and correction method based on simultaneous separation of excitation spectrum and emission spectrum

A technology of emission spectroscopy and quantitative detection, which is applied in the field of fluorescence resonance energy transfer quantitative detection, and can solve the problems of large FRET efficiency value and so on.

Active Publication Date: 2017-05-24
师大瑞利光电科技(清远)有限公司
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Problems solved by technology

Our measurement results show that the FRET efficiency measured by the ExEm-spFRET method is generally too large

Method used

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  • FRET (Fluorescence Resonance Energy Transfer) quantitative detection and correction method based on simultaneous separation of excitation spectrum and emission spectrum
  • FRET (Fluorescence Resonance Energy Transfer) quantitative detection and correction method based on simultaneous separation of excitation spectrum and emission spectrum
  • FRET (Fluorescence Resonance Energy Transfer) quantitative detection and correction method based on simultaneous separation of excitation spectrum and emission spectrum

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Embodiment 1

[0054] 1. Plasmid source

[0055] The donor fluorescent group is the gene-encoded fluorescent protein Cerulean (referred to as C), and the acceptor is the gene-encoded fluorescent protein Venus (referred to as V). Referring to the tandem C32V plasmid, it is composed of a 32-amino acid link sequence (TSGLETRDIRSENLYFQGPREFPGGTAGPVAT) that links C and V. , with reference to the tandem CTV plasmid that is Cerulean-TRAF-Venus, wherein TRAF is a receptor-associated factor domain of a long-chain tumor necrosis factor comprising 229 amino acids; the tandem plasmid CVC (Cerulean-5-Venus-5-Cerulean) to be tested contains Two donors C and one acceptor V, C and V are connected by 5 amino acid sequences, these plasmids were purchased from the US addgene plasmid bank [Koushik S V, Blank P S, Vogel S S. Anomalous surplus energy transfer observed with multiple FRET acceptors[J].PloS one,2009,4(11):e8031].

[0056] 2. Wide-field spectral microscopy imaging system

[0057] The wide-field flu...

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Abstract

The invention discloses an FRET (Fluorescence Resonance Energy Transfer) quantitative detection and correction method based on simultaneous separation of an excitation spectrum and an emission spectrum, and belongs to the technical field of FRET quantitative detection. The FRET quantitative detection and correction method comprises the following steps of measuring an excitation-emission spectrum of a reference sample; carrying out linear separation on the excitation-emission spectrum of the reference sample according to SD, SA and SS, thus obtaining three weight factors; computing system correction factors for ExEm-spFRET quantitative detection by utilizing the three weight factors, and then using the system correction factors for measuring the apparent FRET efficiency of a to-be-detected sample. An ExEm-spFRET method corrected by utilizing the obtained system correction factors is an m-ExEm-spFRET method, the measurement result is more accurate, and the ExEm-spFRET method is suitable for different detection systems; an application range of the ExEm-spFRET method can be greatly promoted, so that the application range of an FRET detection technology in cytobiology is increased.

Description

technical field [0001] The invention belongs to the technical field of fluorescence resonance energy transfer (Fluorescence Resonance Energy Transfer, FRET) quantitative detection, and specifically relates to a FRET quantitative detection (ExEm- spFRET) correction method. Background technique [0002] Fluorescent protein-based FRET technology has emerged as a powerful tool for studying fundamental biochemical events in living cells. Quantitative FRET detection is an inevitable requirement for academic exchanges and comparison of test results from different laboratories. FRET efficiency (E, the ratio of the energy transferred from the donor to the acceptor to the total energy absorbed by the donor) is an important indicator for the quantification of FRET. The occurrence of FRET requires a large overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor. Since the fluorescent protein excitation spectrum has a long tail in the short wave, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486G01N2201/121
Inventor 陈同生杜孟艳
Owner 师大瑞利光电科技(清远)有限公司
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