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Method for identifying quantity of cysteine in protein and application thereof

A cysteine ​​residue and protein technology, applied in the field of identifying the number of cysteine ​​in proteins, to achieve the effect of improving efficiency

Inactive Publication Date: 2013-11-20
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Detecting the number of cysteine ​​residues plays a very important role in the discovery of high-quality subunits and the improvement of wheat quality, but there is still a lack of rapid and efficient methods for identifying the number of cysteine ​​in wheat high molecular weight glutenin subunits at home and abroad

Method used

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  • Method for identifying quantity of cysteine in protein and application thereof
  • Method for identifying quantity of cysteine in protein and application thereof
  • Method for identifying quantity of cysteine in protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1, identification of the number of cysteine ​​residues in wheat high molecular weight glutenin subunits

[0027] 1. Extraction and sample preparation of wheat high molecular weight glutenin subunits

[0028] Take two portions of 15 mg of wheat Bumper flour, put them into 1.5 ml centrifuge tubes respectively; add 1 ml of 70% ethanol by volume, vortex for 30 min, centrifuge at 12000 rpm for 10 min, and remove the supernatant. Then add 1 ml of isopropanol with a volume percentage of 55% respectively, bathe in water at 65° C. for 30 min, centrifuge at 12000 g for 10 min, and remove the supernatant. Then the remaining supernatant was blotted dry with filter paper, and the above steps were repeated 3 times. Then add 0.1ml of DTT-containing solution A (the mass percentage of DTT and solution A is 1:100), wherein the composition of solution A is, isopropanol: 1M Tris-HCl (pH8): double distilled water=50:8: 42 (volume ratio); vortex to mix, 65 ° C water bath for 30 min...

Embodiment 2

[0035] Embodiment 2, utilize the wheat variety that 17 known subunits are composed to verify the method of the present invention

[0036] 1. Extraction and sample preparation of wheat high molecular weight glutenin subunits

[0037] Take Ajana, Banks, Bullaring, Bumper, Calingiri, Chara, EGA Blanco, Endure, IGW2944, IGW3240, Pugsley, M12, M25, M26, Shan229, Wanmai33, Halberd and other 17 different varieties of wheat seeds, sampling and sample preparation and mass spectrometry Identification is equivalent to Example 1.

[0038] The mass spectrometry results of wheat variety Shan229 are shown in Figure 2. in, Figure 2A Mass spectrum of high molecular weight glutenin subunits in common wheat variety Shan229 treated with 4-vinylpyridine, Figure 2B It is the mass spectrum of high molecular weight glutenin subunits in common wheat variety Shan229 without 4-vinylpyridine treatment.

[0039] The mass spectrometry results of 17 different wheat varieties are shown in Table 1 and T...

Embodiment 3

[0046] Example 3, using the number of cysteine ​​residues in salt-soluble protein and water-soluble protein in lupine to verify the method of the present invention

[0047] 1. Sample extraction and preparation

[0048] Salt-soluble protein: Take two 15mg lupine powders, add 0.2ml of 0.5M NaCl solution to one of the samples, vortex for 30 minutes, and then add 0.2ml of 0.5M containing 4-vinylpyridine to the other sample M NaCl solution (volume percent of 4-vinylpyridine and 0.5M NaCl solution is 1.4:98.6), vortexed, 30min in a water bath at 65°C, and centrifuged at 12000rpm for 10min; Acetone precipitated overnight at room temperature; after overnight precipitation, both samples were centrifuged at 12000rpm for 10min, the supernatant was discarded, the precipitate was dried for 10min, and 70 μl of chromatographic grade acetonitrile solution (which contained TFA with a final concentration of 0.1%) was added, dissolved for more than 4 hours, and the obtained The supernatant was ...

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Abstract

The invention discloses a method for identifying quantity of cysteine in protein, especially a method for identifying quantity of cysteine in high molecular weight wheat glutenin subunits. The method enables composition of high molecular weight wheat glutenin subunits and alleles to be identified rapidly and accurately and quantity of cysteine residues in each high molecular weight glutenin subunits to be identified. The method provided in the invention is applicable to improvement of wheat quality and rapid detection of the quantity of cysteine residues in high molecular weight glutenin subunits, thereby enabling high-quality subunits to be explored and screened and efficiency of quality improvement to be improved. Furthermore, the method can also be used for identifying quantity of cysteine residues in salt-soluble protein and water-soluble protein of lupin, and therefore, a complete technological system for identification of quantity of cysteine residues is formed.

Description

technical field [0001] The present invention relates to a method for identifying the amount of cysteine ​​in protein and its application, in particular to a method for quickly identifying the amount of cysteine ​​in wheat high molecular weight glutenin subunits and its application in wheat quality improvement . Background technique [0002] Wheat is one of the three most important food crops in the world, and its output ranks second only to corn. my country's wheat planting area, total output and consumption rank first in the world. Wheat seeds are rich in nutrition, rich in starch, protein, fat, minerals, calcium, iron, thiamine, riboflavin, niacin and vitamin A, etc., which meet the physiological needs of the human body and are very beneficial to human health. About 35% of the world's population takes wheat as a staple food, and wheat protein is an important source of plant protein for humans, accounting for about 38.4% of grain protein; Burning, sesame seed cakes, panca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/62G01N1/28G01N1/38C07K1/14
Inventor 晏月明王轲李小辉马武军
Owner CAPITAL NORMAL UNIVERSITY
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