Method for detecting concentration of protein in liquid

A protein concentration and detection method technology, applied in the field of detection and food inspection, can solve the problem of not having the same or similar structure and mechanism of action, and achieve the effect of economical and practical interference, high sensitivity and low interference.

Inactive Publication Date: 2012-07-04
侯巍 +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although various peroxidases in nature have common biological activities, they do not have the same or similar structure and mechanism of action.

Method used

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  • Method for detecting concentration of protein in liquid
  • Method for detecting concentration of protein in liquid
  • Method for detecting concentration of protein in liquid

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Experimental program
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Embodiment 1

[0052] (1) Experimental reagents:

[0053] 1. Substrate buffer: 0.2M Na 2 HPO 4 25.7ml, 0.1M citric acid 24.3ml, add water to 50ml.

[0054] 2. TMB (tetramethylbenzidine) use solution: TMB (10mg / ml absolute ethanol), 0.5ml substrate buffer (PH5.5) 10ml, 0.75% H 2 o 2 32 μl.

[0055] 3. PH 7.4 PBS: KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, NaCl 8.0g, KCl 0.2g, add water to 1L.

[0056] 4. Termination solution: 178.3 ml of distilled water, 21.7 ml of concentrated sulfuric acid (98%) was added dropwise.

[0057] 5. Luminol solution: Weigh a certain amount of luminol and add it to PBS, then put it into the ultrasonic generator for 10 seconds, take it out, add 1 drop of triethylamine to help dissolve it, filter it and use it. Luminol was diluted 100 times with PBS, and hydrogen peroxide was added to a final concentration of 100mM.

[0058] (2) Experimental method:

[0059] 1. Experimental steps using TMB as substrate:

[0060] Dilute hypoheme (or hypoheme hexapepti...

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Abstract

The invention discloses a method for detecting concentration of protein in a liquid, and belongs to the technical field of food inspection and detection. The method comprises the following steps that: the protein in a liquid sample is combined with ferric porphyrin to form a compound with a peroxidase function, the compound catalyzes hydrogen peroxide (H2O2) to generate active oxygen, and the active oxygen oxidizes luminol to form an organic peroxide; the peroxide is instable, nitrogen is immediately obtained through decomposition, and excited state amino-o-phthalic acid is generated; and during conversion from an excited state to a ground state, the released energy is detected in a photon form, and the quantity of the protein in the liquid sample to be detected can be calculated by detecting the change of the number of photons before and after reaction. The invention has the advantages that: the method has the characteristics of high sensitivity, low interference, economy, practicability and the like, and can be widely applied to the field of food safety monitoring in medicine health and food manufacturers, farmer's markets, quality supervision and epidemic prevention departments and the like.

Description

technical field [0001] The invention belongs to the technical field of food inspection and detection. Background technique [0002] Protein is closely related to the origin, existence and evolution of life, and the determination of protein involves many fields such as life science and food science. At present, the protein determination mainly includes Kjeldahl method, isoelectric point precipitation method, ultraviolet absorption method, biuret method, Coomassie brilliant blue staining method, capillary electrophoresis method, ELISA method, direct potential method, high performance liquid chromatography detection, etc. There are three classical methods commonly used, namely Kjeldahl method, double urea method and ultraviolet absorption method. In addition, there is a new assay method that has been widely used in the past ten years, that is, the Coomassie brilliant blue method. Among them, the Coomassie brilliant blue method has the highest sensitivity. Although the nitrog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 王丽萍侯巍郭常润侯玥
Owner 侯巍
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