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Method for extracting total plant proteins and special extraction liquid thereof

A technology of plant total protein and extraction method, applied in the field of fast and efficient protein extraction, can solve the problems of protein loss, incomplete extraction, slow speed, etc., and achieve the effects of wide distribution area, good solubility, and clear detection bands

Inactive Publication Date: 2018-09-07
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The TCA / acetone precipitation method is a commonly used plant protein separation method, which can effectively inhibit the activity of various enzymes in the sample and remove the interference of secondary metabolites such as phenols. Slow speed and incomplete extraction often result in protein loss

Method used

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  • Method for extracting total plant proteins and special extraction liquid thereof
  • Method for extracting total plant proteins and special extraction liquid thereof
  • Method for extracting total plant proteins and special extraction liquid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, application of the present invention extracts tobacco leaf total protein

[0026] (1) Three parts of 1 g tobacco leaves were thoroughly ground and pulverized in liquid nitrogen in sequence, and 10 ml of pre-cooled 10% TCA / acetone (containing 20 µM β-mercaptoethanol) solution was added, vortexed to disperse the tissue powder evenly, and placed -20°C overnight. Take out and centrifuge at 10000g for 30 minutes at 4°C, and discard the supernatant. Then the precipitate was fully washed with pre-cooled acetone (containing 20 µM β-mercaptoethanol), the supernatant was discarded, and the washing was repeated 3 times, and the precipitate was dried at room temperature to obtain a dry protein powder.

[0027] (2) Extract the total protein with the protein dry powder extract of the present invention, add 1 ml of the extract to 100 mg protein dry powder, let stand at room temperature for 5 minutes, let the protein dry powder fully absorb and dissolve the protein, and ...

Embodiment 2

[0030] Embodiment 2, applying the present invention to extract the total protein of leaves of crape myrtle mutant and wild type

[0031] (1) Take 1 g of three replicate samples of leaves of Lagerstroemia myrtle mutant and wild type, grind them thoroughly in liquid nitrogen, add 10 ml of pre-cooled 10% TCA / acetone (containing 20 µM β-mercaptoethanol) solution , vortex to disperse the tissue powder evenly, and let stand overnight at -20°C. Take out and centrifuge at 10000g for 30 minutes at 4°C, and discard the supernatant. The precipitate was fully washed with pre-cooled acetone (containing 20 µM β-mercaptoethanol), the supernatant was discarded, and the washing was repeated 3 times, and the precipitate was dried at room temperature to obtain a dry protein powder.

[0032] (2) Extract the total protein with the protein dry powder extract of the present invention, add 1 ml of the extract to 100 mg protein dry powder, let stand at room temperature for 5 minutes, let the protein ...

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Abstract

The invention discloses a method for extracting total plant proteins and a special extraction liquid thereof. A dry protein powder is prepared from plant leaves by a TCA / acetone precipitation method,and then the total proteins in the dry powder are extracted by the special extraction liquid. The special protein extraction liquid is an aqueous solution and is prepared from the following raw materials: 25 mM HEPES, 150 mM KCl, 2 mM MgCl2, 9 M urea, 1% Chaps, 1 mM DTT, 1 mM PMSF, and a protease inhibitor. The extraction liquid can quickly and efficiently extract the total proteins from the dry protein powder precipitated by TCA / acetone, the limitation of the dry protein powder solubility by the TCA / acetone method is avoided, and the obtained total proteins has the advantages of clear band, wide distribution area, no tailing and no dispersion by SDS-PAGE analysis, the protein composition of a research object can be relatively comprehensively reflected, and the plant total proteins are fast and efficiently extracted. The method has the advantages of short extraction time, good reproducibility and no impurity interference of the extracted proteins, and can satisfy the proteomic analysisof LC-MS.

Description

technical field [0001] The invention relates to a method for extracting total plant protein in the field of proteomics research, in particular to extracting protein quickly and efficiently from protein dry powder precipitated by TCA / acetone. Background technique [0002] In recent years, the rapid development of proteomics-related technologies has pushed the application of proteomics to almost all biological fields, including botany. The preparation of protein samples is the basis of proteomics research, which directly affects the later stage of omics analysis. The ideal sample preparation method should be able to extract all the proteins in the sample to the maximum extent, and at the same time have good repeatability. Therefore, the sample preparation follows the following principles: use as simple a method as possible for sample processing to avoid protein loss; minimize protein degradation; and maximize protein solubility. Plant tissues usually have relatively low prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
CPCC07K1/145
Inventor 李素梅李亚王淑安王鹏杨如同
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI