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A kind of acetyl coenzyme A content determination kit and method thereof

A technology for the determination of acetyl-CoA, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of low content of acetyl-CoA, difficulty in detection, and low sensitivity, so as to simplify the detection steps and shorten the reaction time. , the effect of easy operation

Active Publication Date: 2016-08-17
SUZHOU COMIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The original acetyl-CoA content detection method has low sensitivity, and it is usually difficult to detect due to the low content of acetyl-CoA in the sample and a lot of interference
At present, there is no easy-to-operate, fast and sensitive kit on the market, which allows the tester to easily determine the content of acetyl-CoA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] A kit for measuring acetyl-CoA content, comprising the following reagents:

[0067] Reagent 1, in liquid form, stored at 4°C, consists of polyvinylpyrrolidone (PVP) with a mass of 2.5g, mercaptoethanol with a volume of 37uL, a concentration of 100mM, a volume of phenylmethylsulfonyl fluoride (PMSF) with a volume of 500uL and a volume of Dissolve 5mL of glycerol in Tris-HCl buffer solution with a concentration of 50mM, pH=7.5, and a volume of 100mL, and place it in a reagent bottle;

[0068] Reagent 2, in powder form, stored at -20°C, composed of 0.3 mg of malate dehydrogenase (MDH), placed in a 1.5mLEP tube;

[0069] Reagent 3, in liquid form, stored at 4°C, composed of citrate synthase with a volume of 10uL, placed in a 1.5mLEP tube;

[0070] Reagent 4, in powder form, stored at -20°C, composed of 30 mg of malic acid and 7.5 mg of oxidized coenzyme I (NAD), placed in a 30 mL reagent bottle;

[0071] Reagent 5, in liquid form, stored at 4°C, consists of a Tris-HCl buf...

Embodiment 2

[0104] A kit for measuring acetyl-CoA content, comprising the following reagents:

[0105] Reagent 1, in liquid form, stored at 4°C, consists of polyvinylpyrrolidone (PVP) with a mass of 2.5g, mercaptoethanol with a volume of 37uL, a concentration of 100mM, a volume of phenylmethylsulfonyl fluoride (PMSF) with a volume of 500uL and a volume of Dissolve 5mL of glycerol in Tris-HCl buffer solution with a concentration of 50mM, pH=7.5, and a volume of 100mL, and place it in a reagent bottle;

[0106] Reagent 2, in powder form, stored at -20°C, composed of 0.3 mg of malate dehydrogenase (MDH), placed in a 1.5mLEP tube;

[0107] Reagent 3, in liquid form, stored at 4°C, composed of citrate synthase with a volume of 10uL, placed in a 1.5mLEP tube;

[0108] Reagent 4, in powder form, stored at -20°C, composed of 30 mg of malic acid and 7.5 mg of oxidized coenzyme I (NAD), placed in a 30 mL reagent bottle;

[0109] Reagent 5, in liquid form, stored at 4°C, consists of a Tris-HCl buf...

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PUM

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Abstract

The invention discloses a kit and a method for content determination of acetyl coenzyme A. The kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V, wherein the reagent I is prepared from Tris-HCl, polyvinylpyrrolidone, mercaptoethanol, phenylmethylsulfonyl fluoride and glycerinum; the reagent II is prepared from malic dehydrogenase; the reagent III is prepared from citrate synthase; the reagent IV is prepared from malic acid and an oxidation type coenzyme I; and the reagent V is prepared from Tris-HCl. The malic dehydrogenase can be used for catalyzing malic acid and the oxidation type coenzyme I to generate oxaloacetic acid and a reduced coenzyme I; the citrate synthase is used for catalyzing acetyl coenzyme A and oxaloacetic acid to generate citric acid and a coenzyme A; by virtue of a coupled reaction of the malic dehydrogenase and the citrate synthase, the content of the acetyl coenzyme A and the generation rate of the reduced coenzyme I are in a direct proportion; the climbing speed of the light absorption value at 340nm reflects the content of the acetyl coenzyme A. The kit disclosed by the invention is simple and convenient to operate, high in detection sensitivity and high in recovery rate, and the cost of the reagents is remarkably lowered. The detection steps are further simplified, so that the kit is simpler and more convenient and efficient to test.

Description

technical field [0001] The invention relates to the field of kits, in particular to a kit for measuring acetyl-CoA content and a method thereof. Background technique [0002] Acetyl-CoA widely exists in animals, plants, microorganisms and cultured cells, and is an important intermediate metabolite produced during the metabolism of energy substances in organisms. Acetyl-CoA is a pivotal substance in the metabolism of energy substances in the body. The three major nutrients of sugar, fat and protein converge into a common metabolic pathway through acetyl coenzyme A - tricarboxylic acid cycle and oxidative phosphorylation. Through this pathway, carbon dioxide and water are completely oxidized to release energy for the synthesis of ATP. In addition, acetyl-CoA is a precursor for the synthesis of fatty acids, ketone bodies, cholesterol and its derivatives and other physiologically active substances. The original acetyl-CoA content detection method has low sensitivity, and it is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/527C12Q1/32
Inventor 姚金美赵林川
Owner SUZHOU COMIN BIOTECH
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