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Uridine diphosphate glucose pyrophosphorylase activity detecting kit and method

A technology of phosphorylase activity and uridine diphosphate, which is applied in the field of life sciences, can solve the problems of inconvenient and quick operation, cumbersome extraction methods, and large measurement system, and achieve sensitive and reliable detection results, small amount of measurement reagents, and high measurement system small effect

Inactive Publication Date: 2016-04-13
SUZHOU COMIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the determination method of uridine diphosphate glucose pyrophosphorylase is mainly the enzyme coupling method, and the extraction methods are not the same. The extraction methods reported in many literatures are cumbersome, the components are complex, the measurement system is large, and the operation is not simple and fast enough.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Step 1 preparation of instruments and supplies;

[0075] Balance, cryogenic centrifuge, mortar, UV spectrophotometer, 1mL quartz cuvette;

[0076] The preparation of step 2 reagent;

[0077] The reagent two is fully dissolved with 5 mL of distilled water before use to make a reagent two aqueous solution;

[0078] The reagent three is fully dissolved with 5 mL of distilled water before use to make an aqueous solution of reagent three;

[0079] The reagent four is fully dissolved with 5 mL of distilled water before use to make an aqueous solution of reagent four;

[0080] Extraction of crude enzyme solution in the tissue sample in step 3;

[0081] Add the extract according to the ratio of mass (g): extract volume (mL) of 1: (5~10), homogenize in ice bath, centrifuge at 12000rpm for 10min at 4°C, take the supernatant, that is, the crude enzyme solution, and placed on ice for testing;

[0082] Step 4 Determination of uridine diphosphate glucose pyrophosphorylase activi...

Embodiment 2

[0095] Step 1 preparation of instruments and supplies;

[0096] Balance, cryogenic centrifuge, mortar, UV spectrophotometer, 1mL quartz cuvette;

[0097] The preparation of step 2 reagent;

[0098] The reagent two is fully dissolved with 5 mL of distilled water before use to make a reagent two aqueous solution;

[0099] The reagent three is fully dissolved with 5 mL of distilled water before use to make an aqueous solution of reagent three;

[0100] The reagent four is fully dissolved with 5 mL of distilled water before use to make an aqueous solution of reagent four;

[0101] Extraction of crude enzyme solution in the tissue sample in step 3;

[0102] Add the extract according to the ratio of mass (g): extract volume (mL) of 1: (5~10), homogenize in ice bath, centrifuge at 12000rpm for 10min at 4°C, take the supernatant, that is, the crude enzyme solution, and placed on ice for testing;

[0103] Step 4 Determination of uridine diphosphate glucose pyrophosphorylase activi...

Embodiment 3

[0117] Step 1 preparation of instruments and supplies;

[0118] Balance, cryogenic centrifuge, mortar, UV spectrophotometer, 1mL quartz cuvette;

[0119] The preparation of step 2 reagent;

[0120] The reagent two is fully dissolved with 5 mL of distilled water before use to make a reagent two aqueous solution;

[0121] The reagent three is fully dissolved with 5 mL of distilled water before use to make an aqueous solution of reagent three;

[0122] The reagent four is fully dissolved with 5 mL of distilled water before use to make an aqueous solution of reagent four;

[0123] Extraction of the crude enzyme solution in the cell sample in step 3;

[0124] According to the number of cells (10 4 Each): the volume of extract (mL) is (500~1000): 1 ratio, and the cells are ultrasonically disrupted in an ice bath (power 300w, ultrasound for 3 seconds, interval of 7 seconds, total time 3min), and then centrifuged at 4°C, 12000rpm for 10min, Take the supernatant, that is, the crud...

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Abstract

The invention discloses a uridine diphosphate glucose pyrophosphorylase activity detecting kit and method. The kit comprises an extracting solution, a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V, wherein the extracting solution is prepared by uniformly mixing Tris, distilled water, HCl, ethylenediamine tetraacetic acid disodium salt, DTT and saccharose; the reagent I is prepared by uniformly mixing the extracting solution, distilled water, MgCl2 and UDPG; the reagent II is composed of NADP; the reagent III is composed of glucose 6-phosphate dehydrogenase; the reagent IV is composed of phosphoglucomutase; the reagent V is prepared by dissolving PPi into distilled water. According to the kit, lots of groping and adjusting experiments are conducted on the extracting solution components in the uridine diphosphate glucose pyrophosphorylase activity detecting method and a detecting system, and the effects that the extracting solution components are simple, the extracting effect is good, the detecting system is small and the operation is simple, convenient and quick are achieved finally.

Description

technical field [0001] The invention belongs to the field of life sciences and relates to a kit, in particular to a kit for measuring the activity of uridine diphosphate glucose pyrophosphorylase and a method thereof. Background technique [0002] UDP-glucose pyrophosphorylase is a key enzyme in the process of glycogen synthesis in organisms. Catalyzes the activation of glucose before the synthesis of glycogen from glucose, and synthesizes glucose-1-phosphate and UTP molecules into UDP-glucose (UDPG). [0003] At present, the determination method of uridine diphosphate glucose pyrophosphorylase is mainly the enzyme coupling method, and the extraction methods are not the same, and the extraction methods reported in many literatures are cumbersome, the components are complex, the measurement system is large, and the operation is not simple and fast enough. Now it is necessary to specially develop a kit that can easily, quickly and accurately measure uridine diphosphate glucos...

Claims

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Application Information

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IPC IPC(8): C12Q1/533C12Q1/48C12Q1/32
CPCC12Q1/32C12Q1/485C12Q1/533G01N2333/91245
Inventor 赵林川
Owner SUZHOU COMIN BIOTECH
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