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Kit for determining content of coenzyme A based on micro method and method thereof

A determination method and micro-method technology, applied in the field of life science, can solve the problems of inapplicable coenzyme A detection, long detection time of a single sample, and high requirements for instruments and equipment, so as to reduce the amount of reagents, save the detection time, and improve the detection sensitivity. Effect

Inactive Publication Date: 2019-01-15
SUZHOU COMIN BIOTECH
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Problems solved by technology

[0003] At present, the existing coenzyme A content determination method on the market is high performance liquid chromatography. The disadvantages of this method are: 1. The requirements for equipment are relatively high. Higher, 3. It is not suitable for the detection of coenzyme A in various samples such as animals, plants and microorganisms. 4. The detection time of a single sample is too long. 5. The minimum detection limit is not low enough

Method used

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Embodiment Construction

[0065] The present invention will be described in detail below in conjunction with embodiments.

[0066] A coenzyme A content assay kit based on the micro-method, comprising the following reagents:

[0067] Reagent 1, liquid 100mL×1 bottle, composed of 100mL PH7.5 50mM Tris-HCl buffer solution, 74uL mercaptoethanol, 1mL 100mM PMSF and 5mL glycerin, placed in a 100mL volumetric flask, stored at 4°C;

[0068] Reagent 2, liquid 20mL×1 bottle, composed of 16.2mL pH 7.4 50mM Tris-HCl buffer solution and 3.8mL water, placed in a 20mL volumetric flask, stored at 4°C;

[0069] Reagent 3, powder x 1 bottle, composed of 4.2mg MgCl 2 , 3mg ketoglutaric acid, 4mg rotenone and 2mg TPP, placed in a 1.5mL EP tube, stored at -20°C;

[0070] Reagent 4, powder × 1 tube, composed of 23mg NAD, placed in a 1.5mL EP tube, stored at -20°C;

[0071] Reagent 5, powder × 1 tube, composed of 0.4mg 2500U / mg α-ketoglutarate dehydrogenase, placed in a 1.5mL EP tube, stored at -20°C.

[0072] A method f...

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Abstract

The invention discloses a kit for determining a content of a coenzyme A based on a micro method and a method thereof. The kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V, wherein the reagent I is composed of Tris-HCl, mercaptoethanol, PMSF and glycerin; the reagent II is composed of Tris-HCl and water; the reagent III is composed of MgCl2, ketoglutaric acid, rotenone and TPP; the reagent IV is composed of NAD; and the reagent V is composed of alpha-ketoglutaric dehydrogenase. The principle of the method is that alpha-ketoglutaric dehydrogenase can catalyzethe coenzyme A, alpha-ketoglutaric acid and NAD+ to produce succinyl-CoA, carbon hydrate and NADH; the NADH has a characteristic absorption peak at 340 nm; alpha-KGDH, alpha-ketoglutaric acid and NAD+are added in a reaction system; and the content of the coenzyme A is in direct proportion to the production rate of NADH. The kit for determining the content of the coenzyme A based on a micro methodand the method thereof in the invention have the advantages of wide application range, high detection sensitivity, short detection time, less use amount of the reagent and extremely low cost.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a coenzyme A content determination kit based on a trace method and a method thereof. Background technique [0002] Coenzyme A is the coenzyme of transacylase and α-ketoglutarate dehydrogenase in organisms, and plays an important role in various biochemical reactions such as the decomposition of sugar, the synthesis and decomposition of fatty acids, and the metabolism of amino acids. Coenzyme A is mainly used clinically for the adjuvant treatment of diseases caused by abnormal lipid metabolism and other diseases. [0003] At present, the existing coenzyme A content determination method on the market is high performance liquid chromatography. The disadvantages of this method are: 1. The requirements for equipment are relatively high. High, 3. It is not suitable for the detection of coenzyme A in various samples such as animals, plants and microorganisms. 4. The detection t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33G01N21/3577
CPCG01N21/33G01N21/3577
Inventor 姚金美赵林川
Owner SUZHOU COMIN BIOTECH
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