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Method for measuring an excitation emission spectrum linear separation quantitative FRET system correction factor based on same system cell sample and application

A technology of emission spectrum and correction factor, which is applied in the field of fluorescence resonance energy transfer detection, can solve the problems of cumbersome operation, inability to guarantee contrast and background signal, etc., so as to improve the scope of application, improve intelligence and accuracy, QA/QD and KA /KD accurate effect

Active Publication Date: 2021-06-29
师大瑞利光电科技(清远)有限公司 +1
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Problems solved by technology

However, this method needs to switch multiple dishes of cell samples, which is cumbersome to operate, and the same contrast and background signal cannot be guaranteed when four dishes of cell samples are imaged separately. method is of great importance

Method used

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  • Method for measuring an excitation emission spectrum linear separation quantitative FRET system correction factor based on same system cell sample and application
  • Method for measuring an excitation emission spectrum linear separation quantitative FRET system correction factor based on same system cell sample and application
  • Method for measuring an excitation emission spectrum linear separation quantitative FRET system correction factor based on same system cell sample and application

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Embodiment 1

[0078] 1. Plasmid

[0079] A given donor-receptor pair: the donor is a gene-encoded fluorescent protein Cerulean (referred to as C), and the acceptor is a gene-encoded fluorescent protein Venus (referred to as V);

[0080] FRET tandem plasmid structure C5V: FRET tandem plasmid structure composed of 5 amino acid linker sequences linking C and V;

[0081] FRET tandem plasmid structure C17V: FRET tandem plasmid structure composed of 17 amino acid linker sequences linking C and V;

[0082] FRET tandem plasmid structure C32V: FRET tandem plasmid structure composed of 32 amino acid linker sequences linking C and V;

[0083] FRET tandem plasmid structure CTV (Cerulean-TRAF-Venus): a FRET tandem plasmid structure composed of a 229-amino acid linker sequence linking C and V; where TRAF is a long-chain tumor necrosis factor receptor related to 229 amino acids factor field;

[0084] Source: Donor plasmid Cerulean (C), acceptor plasmid Venus (V), and FRET reference plasmids C5V, C17V, C3...

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Abstract

The invention discloses a method for measuring an excitation emission spectrum linear separation quantitative FRET system correction factor based on a same system cell sample and application. The method comprises the following steps: in the same system (such as a vessel of cells), obtaining weight factor graphs Wd, Wa and Ws by utilizing ExEm-spFRET data, and then determining Ws / Wa and Ws / Wd ranges RSA and RSD of different FRET plasmids for independently transfecting the cells; and making a data screening template for the imaging view by using the range, performing data screening, fitting a straight line, and obtaining correction factors KA / KD and QA / QD of the ExEm-spFRET system. According to the method disclosed by the invention, the measurement of the correction factor can be completed by only one vessel of cell sample, the problem that different contrast ratios and background signals exist in independent measurement of different FRET plasmids is avoided, the accuracy of the correction parameter of the measurement system is improved, the use threshold is reduced, and the efficiency is improved.

Description

technical field [0001] The invention belongs to the technical field of fluorescence resonance energy transfer (FRET) detection, and in particular relates to a method and application for measuring excitation emission spectrum linear separation and quantifying FRET system correction factors based on cell samples of the same system. Background technique [0002] Fluorescent proteins (FPs)-based FRET microscopy has become an important tool for studying chemical molecular dynamics in living cells. Obtaining quantitative FRET signals that do not depend on the detection system and the expression level of FPs is an inevitable requirement for academic communication. Recently, we have developed a quantitative FRET measurement technique based on the simultaneous separation of excitation-emission spectra (ExEm-spFRET), which can simultaneously overcome acceptor excitation crosstalk and donor emission crosstalk [Mengyan Du, et al. "Wide-field microscopic FRET imaging using simultaneous ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6458G01N21/6402
Inventor 陈同生孙晗庄正飞
Owner 师大瑞利光电科技(清远)有限公司
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