Method for detecting N-acetyl-beta-D-glucosaminidase electrophoresis reactive dye

A glucosamine and active dyeing technology, applied in the field of enzyme active dyeing, can solve the problems of high cost, long reaction time, large amount of substrate, etc., and achieve the effects of short time-consuming, simple identification, simple and fast operation

Inactive Publication Date: 2010-03-10
XIAMEN UNIV
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Problems solved by technology

However, there are problems such as large amount of substr

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  • Method for detecting N-acetyl-beta-D-glucosaminidase electrophoresis reactive dye

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Embodiment Construction

[0022] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

[0023] 1) Preparation of materials, including preparation of reaction solution, staining solution and enzyme solution.

[0024] The reaction solution was prepared by adding 0.2ml of 95% ethanol to the substrate 1mg of naphthol AS-BI-N-acetyl-β-D-glucosaminide (SIGMA, N4006) to aid dissolution, and then adding 0.1mol / L phosphoric acid at pH 7.5 The buffer was dissolved to 20ml.

[0025] Prepare the staining solution by dissolving 20mg Fast Blue BB salt in 10ml 0.01mol / L Tris-HCl buffer; the Fast Blue BB salt can be Fluka44670 produced by Xiamen Taijing Biotechnology Co., Ltd., the Tris-HCl buffer The pH is preferably 7.5 at 4°C.

[0026] The enzyme solution was prepared by extracting the NAGase crude enzyme solution of Litopenaeus vannamei with 0.01mol / L Tris-HCl buffer solution pH 7.5.

[0027] 2) active staining, active staining comprises the foll...

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Abstract

The invention provides a method for detecting N-acetyl-beta-D-glucosaminidase electrophoresis reactive dye, relating to the reactive dye of an enzyme. Material preparation comprises preparing reactionliquid, dying liquid and enzyme solution; active dye comprises the following steps: preparing Native-PAGE; sampling extracted NAGase crude enzyme solution of prawns; carrying out low temperature electrophoresis in a chromatographic cupboard; taking glue after electrophoresis is completed, placing the glue being washed by distilled water into the reaction liquid, and washing the glue after reaction by distilled water; placing the glue into the dying solution for dying, then taking out and washing by distilled water, placing the glue in water for reservation, therefore, active strips of NAGasecan be detected rapidly. The method uses a small amount of substrate, adopts different azo dyes, has no need of decoloration, simpler and faster operation, is improvement on a NAGase electrophoresis active dying method, and has important significance on rapidly detecting NAGase.

Description

technical field [0001] The invention relates to an enzyme activity staining, in particular to a detection method for electrophoretic activity staining of N-acetyl-β-D-glucosaminidase. Background technique [0002] As a diagnostic indicator, enzymes are simple and rapid. NAGase is one of the key enzymes in the cascade catalytic degradation pathway of chitin, which widely exists in animals, plants and microorganisms. In crustaceans, NAGase is closely related to food digestion, hatching of larvae, periodic molting, and antibacterial protection; in humans, NAGase is related to diseases such as diabetes, kidney disease, breast cancer, and gastric cancer, and has been used as a biochemical indicator for the diagnosis of these diseases Reference and testing. [0003] At present, the research on the activity analysis system and method of NAGase at home and abroad has become mature and perfect. Substances, under acidic conditions, the ability of enzymes to catalyze the hydrolysis ...

Claims

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Application Information

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IPC IPC(8): G01N1/30C12Q1/34
Inventor 谢晓兰魏晓倩王烨黄乾生陈清西
Owner XIAMEN UNIV
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