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Reagent for detecting activity of soluble epoxide hydrolase (sEH) in tissues or cells

A soluble and oxide-based technology, applied in material excitation analysis, fluorescence/phosphorescence, etc., to achieve fast detection, less sample consumption, and strong operability

Active Publication Date: 2013-11-06
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The products of this kind of compound after being acted by sEH can be excited to fluoresce, but the compound itself cannot be excited to fluoresce, but this method is only used to detect the activity of recombinant purified sEH in vitro

Method used

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  • Reagent for detecting activity of soluble epoxide hydrolase (sEH) in tissues or cells
  • Reagent for detecting activity of soluble epoxide hydrolase (sEH) in tissues or cells
  • Reagent for detecting activity of soluble epoxide hydrolase (sEH) in tissues or cells

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Experimental program
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Effect test

preparation example Construction

[0045] The preparation of the detection reagent is as follows:

[0046] 1. Solution preparation

[0047] 1) Lysis solution: hypotonic solution + phenylmethylsulfonyl fluoride (PMSF)

[0048] The hypotonic solution is: 55-65 mg of trishydroxymethylaminomethane (Tris), magnesium chloride (MgCl 2 ) 15-25 mg, ethylene glycol diethyl ether diamine tetraacetic acid (EGTA) 35-40 mg, ethylenediamine tetraacetic acid (EDTA) 3-5 mg dissolved in water, adjust the pH to 7.0-7.5, and dilute the water to 100ml;

[0049] Hypotonic solutions can also be directly used with deionized water.

[0050] 2) Detection buffer: add bovine serum albumin (BSA) at 0.2mg / ml to 50mM Tris, pH=6.8-7.3, store at 4°C;

[0051] 3) Detection substrate: Epoxy Fluor 7 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 5mM stock solution and stored at -20°C;

[0052] 4) sEH inhibitor: 12-(3-adamantan-1-yl-ureido)-n-dodecanoic acid [12-(3-adamantan-1-yl-ureido)-dodecanoic acid, AUDA], trans -4-[4-(3-Adaman...

Embodiment 1

[0067] 1. Solution preparation

[0068] 1) Lysis solution 5ml: deionized water 4.95ml+100mM PMSF50ul;

[0069] 2) Assay buffer: 50mM Tris+0.2mg / ml BSA, pH 7.0, stored at 4°C;

[0070] 3) Detection substrate: Epoxy Fluor 7 was dissolved in DMSO to prepare a 5mM stock solution, stored at -20°C;

[0071] 4) sEH inhibitor: t-AUCB was dissolved in DMSO to make a 10mM stock solution and stored at -20°C;

[0072] 2. Sample

[0073] 1) The sEH inhibitor tAUCB was dissolved in PEG400 to prepare a 4 mg / ml solution. Eleven 8-week-old C57BL / 6 mice were randomly divided into two groups. One group of 6 mice was given the sEH inhibitor tAUCB and added to the drinking water so that the concentration was 20mg / L. The other group of 5 mice was given the same volume of solvent PEG400 to drinking water. The mice had free access to food and water.

[0074] 2) After 4 days of administration, the mice were anesthetized, blood was taken from the right ventricle, and perfused with normal saline f...

Embodiment 2

[0084] 1. Solution preparation

[0085] 1) Prepare a hypotonic solution first: dissolve 55-65 mg of trishydroxymethylaminomethane, 15-25 mg of magnesium chloride, 35-40 mg of ethylene glycol diethyl ether diamine tetraacetic acid, and 3-5 mg of ethylenediamine tetraacetic acid In water, adjust the pH to 7.0-7.5, and dilute the water to 100ml;

[0086] Take 2.475ml of hypotonic solution and add 25ul of PMSF with a concentration of 100mM to make 5ml of lysate;

[0087] 2) Assay buffer: 50mM Tris+0.2mg / ml BSA, pH 7.0, stored at 4°C;

[0088] 3) Detection substrate: Epoxy Fluor 7 was dissolved in DMSO to prepare a 5mM stock solution, stored at -20°C

[0089] 4) sEH inhibitor: t-AUCB was dissolved in DMSO to make a 10mM stock solution, stored at -20°C

[0090] 2. Sample

[0091] 1) Cell culture and treatment: the human embryonic kidney 293 cell line was spread in a 6cm cell culture dish, the cells were 80% confluent, the medium was changed, and treatment was given. Divided int...

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Abstract

A reagent for detecting activity of soluble epoxide hydrolase (sEH) in tissues or cells consists of lysis solution, detection buffer solution, detection substrate solution and sEH inhibitor solution. The invention also provides a preparation method of the reagent.

Description

technical field [0001] The invention relates to a detection reagent, in particular to a reagent for detecting the activity of soluble epioxidase hydrolase. The reagent can measure the activity of soluble epioxide hydrolase in cells or tissues through fluorescence detection. Background technique [0002] Soluble epioxide hydrolase (Soluble epoxy hydrolase, sEH, also known as EPXH2) widely exists in mammals, and is distributed in many tissues and organs such as liver, kidney, lung, heart, brain, spleen, vascular endothelium and smooth muscle ;sEH protein is a homodimer composed of two monomers with a size of 62kD. The N-terminal and C-terminal of each monomer have different enzymatic activities, the N-terminal has phosphatase activity, and the C-terminal has epioxidation Hydrolase activity, which can hydrolyze epioxidized fatty acids or other epioxides and convert them into corresponding diols, thereby reducing the chemical reactivity of epioxides, increasing their water solub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 朱毅刘燕何金龙李楠
Owner PEKING UNIV
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