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A method for detecting the binding ability of a sample to be tested and gpr40 and its special specific fluorescent probe

A fluorescent probe and binding ability technology, applied in the field of biological analysis, can solve problems such as research on the effect and mechanism of difficult compounds, unclear binding sites, inactivation of receptor proteins, etc., and achieve simple preparation methods, simple operations, Good binding effect

Inactive Publication Date: 2018-11-13
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent probes are not harmful to radioactive probes, but this method also needs to separate and extract the GPR40 protein from the cell membrane, which may easily cause the inactivation of the receptor protein. At the same time, an immune reaction is required to immobilize GPR40 on the magnetic beads, and the operation is more complicated.
Although the radioactive probes and fluorescent probes developed by the above researchers can bind to GPR40, their binding sites on GPR40 are unknown. Clear, it is difficult to conduct in-depth research on the effect and mechanism of the compound

Method used

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  • A method for detecting the binding ability of a sample to be tested and gpr40 and its special specific fluorescent probe
  • A method for detecting the binding ability of a sample to be tested and gpr40 and its special specific fluorescent probe
  • A method for detecting the binding ability of a sample to be tested and gpr40 and its special specific fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the preparation of fluorescent probe F-TAK-875A

[0067] The chemical reaction flow chart for preparing fluorescent probe F-TAK-875A is shown in figure 1 . Prepare the fluorescent probe F-TAK-875A as follows:

[0068] 1. Preparation of Compound 1

[0069] (1) Take two round bottom flasks, first add 4-bromo-3,5-xylenol (see figure 1 ⑴) 6g, 3-formylphenylboronic acid (see figure 1 (2) 5g, 1M Na in 2 CO 3 80 mL of aqueous solution, 40 mL of ethanol and 80 mL of toluene were added, and 1.8 g of tetrakis(triphenylphosphine)palladium was added, and stirred overnight at room temperature under the protection of argon.

[0070] (2) After completing step (1), first remove the solvent by distillation under reduced pressure, then load the residue to the silica gel chromatography column, then wash the silica gel chromatography column with 5 column volumes of eluent 1 and collect After passing through the column solution, the solution after passing through the co...

Embodiment 2

[0126] Example 2, Application of fluorescent probe F-TAK-875A to detect the binding ability of TAK-875 and GPR40

[0127] 1. HEK293 cells transiently transfected to express GPR40

[0128] ReferenceLipofectamine TM Transient transfection of HEK293 cells according to the operation steps of the 2000 kit. Specific steps are as follows:

[0129] (1) Inoculate HEK293 cells in DMEM medium containing phenol red containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin, at 37°C, 5% CO 2 cultivated in the environment.

[0130] (2) Take a cell culture dish with a diameter of 10 cm, and inoculate the system obtained in step (1) (containing 5×10 6 HEK293 cells), at 37°C, 5% CO 2 The environment was cultured for 12 hours to make HEK293 cells adhere to the wall.

[0131] (3) Take 8 μg of the plasmid expressing GPR40 and dilute it with 0.5 mL of DMEM medium containing phenol red to obtain a plasmid solution.

[0132] (4) Take 20 μL Lipofectamine 2000 (Lipofectamine TM 2000 ...

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Abstract

The invention discloses a method for detecting the binding capacity between a to-be-detected sample and GPR40 and a specific fluorescent probe. The specific fluorescent probe is a compound represented by formula (II), wherein R is an inorganic or organic group capable of generating fluorescence detection signals. An experiment proves that the method can be used for detecting the binding capacity between the to-be-detected sample and the GPR40. The method for detecting the binding capacity between the to-be-detected sample and the GPR40 has the advantages that the bonding site is clear and definite, the specificity is high, the operation is simple, and the specific fluorescent probe is good in stability and has no harm. The method provided by the invention has an important application value in detection of the binding capacity between the to-be-detected sample and the GPR40.

Description

technical field [0001] The invention belongs to the field of biological analysis, and in particular relates to a method for detecting the binding ability of a sample to be tested and GPR40 and a special specific fluorescent probe thereof. Background technique [0002] G protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1 (FFAR1), is a G protein with 7 transmembrane alpha helices Coupled receptors. Studies have found that GPR40 plays an important role in regulating insulin secretion. For example, in pancreatic β cells, GPR40 can be activated by free fatty acids, which can increase the concentration of intracellular calcium ions, thereby promoting insulin release. Therefore, using GPR40 as a drug target molecule for the treatment of diabetes is currently a research hotspot at home and abroad, and a large amount of research work has been devoted to screening drugs that can act on GPR40, but the methods that can be used to quantitatively detect the dir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C09K11/06C07D407/12C07D307/80
CPCC07D307/80C07D407/12C09K11/06C09K2211/1007C09K2211/1088G01N21/6486
Inventor 任肖敏曹林英杨郁郭良宏
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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