Kit and method for measuring content of 1,3-diphosphoglycerate based on micromethod

A technology of diphosphoglycerate and a determination method, which is applied in the field of 1,3-diphosphoglycerate content determination kits, can solve the problems of long detection period, narrow application range, low accuracy, etc., and saves detection time and operation. Simple and accurate results

Inactive Publication Date: 2019-02-12
SUZHOU COMIN BIOTECH
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Problems solved by technology

[0003] At present, the existing method for detecting the content of 1,3-diphosphoglycerate is double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA), but this method has long detection cycle, high cost, ...

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Embodiment Construction

[0070] The present invention will be described in detail below in conjunction with embodiments.

[0071] A 1,3-bisphosphoglycerate content assay kit based on the micromethod, comprising the following reagents:

[0072] Extract solution, liquid 100mL×1 bottle, made by mixing 100mL 100mM pH8.2 Tris-HCl, 38mg EGTA, 50μL mercaptoethanol, 5mL glycerin, and store in a 100mL volumetric flask at 4°C;

[0073] Reagent 1, powder × 1 bottle, composed of 8.4mg cysteine, 8.4mg MgSO 4 .7H 2 O, 2.4mg NADH, 7.2mg ATP, 0.5mg glyceraldehyde triphosphate dehydrogenase mixed, placed in a 30mL reagent bottle, stored at -20°C;

[0074] Reagent 2, liquid 20mL×1 bottle, composed of 84mM pH7.6 Tris-HCl, placed in a 20mL volumetric flask, stored at 4°C.

[0075] A method for measuring the content of 1,3-diphosphoglycerate based on the micro method using the above kit, the specific steps are as follows:

[0076] Step 1 Preparation of instruments and supplies;

[0077] Prepare spectrophotometer or m...

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Abstract

The invention discloses a kit and a method for measuring the content of 1,3-diphosphoglycerate based on a micromethod. A reagent of the kit comprises an extracting solution, a first reagent and a second reagent, wherein the extracting solution is prepared from Tris-HCl, EGTA, mercaptoethanol and glycerinum in a mixed mode, the first reagent is prepared from MgSO4.7H2O, NADH, ATP and glyceraldehyde-3-phosphate dehydrogenase in a mixed mode, and the second reagent is prepared from Tris-HCl. The method comprises the principle that the glyceraldehyde-3-phosphate dehydrogenase can reversely catalyze the 1,3-diphosphoglycerate and the NADH to generate glyceraldehyde-3-phosphate, inorganic phosphorus and NAD, the glyceraldehyde-3-phosphate dehydrogenase and the NADH are added into a reaction system, and by measuring the reduction amount of the NADH at 340 nm, the content of the 1,3-diphosphoglycerate can be reflected. The kit and the method for measuring the content of the 1,3-diphosphoglycerate have the characteristics that the application range is wide, operation is easy and convenient, the detection time is short, detection sensitivity is high, the cost is low, repeatability is good, and the accuracy rate is high.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a 1,3-diphosphoglyceric acid content determination kit and a method based on a trace method. Background technique [0002] 1,3-diphosphoglycerate is an intermediate metabolite in the glycolysis process, which is closely related to the gluconeogenesis pathway, the maintenance of blood sugar concentration in the body and the occurrence of diabetes, and plays an important role in the body's sugar, lipid and protein metabolism disorders. [0003] At present, the existing method for detecting the content of 1,3-diphosphoglycerate is double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA), but this method has long detection cycle, high cost, narrow application range, poor stability and low accuracy And false positives and other shortcomings, which lead to the fact that there is no effective biochemical method to detect the content of 1,3-diphosphoglycerate i...

Claims

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Application Information

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IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 姚金美
Owner SUZHOU COMIN BIOTECH
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