Extraction method of ginkgo leaf chloroplast protein

A technology of Ginkgo biloba leaves and extraction methods, applied in the field of biochemistry, can solve the problems that the efficiency and quality of chloroplast protein extraction are not enough to meet proteomics, the preparation of chloroplast protein samples of Ginkgo biloba leaves is difficult, and the resolubility of protein powder is poor, etc., to achieve focus The effect of good condition, smooth and clear appearance, and little effect of streaks

Inactive Publication Date: 2015-05-06
NANJING FORESTRY UNIV +2
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Problems solved by technology

It can be seen that the preparation of chloroplast protein samples of ginkgo leaves is more difficult
TCA-acetone precipitation is currently the most common method for protein extraction. Although the operation steps are relatively simple, the yield of protein crude extract is relatively high. [5] , but the protein powder obtained by this method has poor resolubility when extracting Ginkgo chloroplast protein, and there are more imp

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  • Extraction method of ginkgo leaf chloroplast protein
  • Extraction method of ginkgo leaf chloroplast protein
  • Extraction method of ginkgo leaf chloroplast protein

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Embodiment Construction

[0031] 1. Materials and methods

[0032] 1.1 Materials

[0033] Ginkgo biloba leaves, collected from the campus of Nanjing Forestry University, Nanjing, Jiangsu Province

[0034] 1.2 Extraction solution and medium

[0035] (1) Chloroplast extraction buffer: 0.3M sorbitol, 1mM MgCl2, 50mM HEPES / KOH (pH 7.8), 2mM EDTA, 0.04% mercaptoethanol, 0.1% PVP.

[0036] (2) Chloroplast isolation buffer: 0.3M sorbitol, 1mM MgCl2, 50mM HEPES / KOH (pH 7.8), 2mM EDTA.

[0037] (3) Percoll density gradient solution: Prepare 80% and 40% Percoll with chloroplast separation buffer respectively, and add 2.5ml 80% and 5ml 40% Percoll in sequence from top to bottom in a 15ml centrifuge to form a secondary density gradient separation solution .

[0038] (4) Protein extraction buffer: 100mM Tris, 100mM EDTA, 50mM borax, 50mM vitamin C, 1% Triton X-100, 2% mercaptoethanol, 30% sucrose.

[0039] (5) Sample treatment solution I (10ml): 2g TCA (trichloroacetic acid), 0.02g DTT, dissolved with acetone ...

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Abstract

The invention belongs to the field of biological chemistry and specifically relates to extraction method of chloroplast protein of xylophyta ginkgo leaf. The method comprises the following steps of: firstly, preparing coarse chloroplast particles from the ginkgo leaves, performing gradient centrifugation through Percoll, so as to obtain complete chloroplast by a specific centrifugal force; then, dissolving protein by using a protein extraction buffer solution, adding the same volume of Tris-balanced phenol for extraction, and precipitating protein by using a ammonium acetate methanol solution. The extraction method is capable of effectively increase extraction efficiency and purity of chloroplast protein. The ginkgo leaf chloroplast protein obtained by the method can be used for proteomics analysis of ginkgo leaf and other xylophyta materials rich in interfering substances, so that solubility and purity of protein are improved; a 2-DE map of the protein has a clear background and many protein spots are distinguishable.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a method for extracting chloroplast protein from leaves of woody plant ginkgo biloba. Background technique [0002] Ginkgo biloba L. is a deciduous tree of the genus Ginkgo in the Ginkgo family. It is known as the "living fossil" in the plant kingdom and is the oldest gymnosperm among the existing seed plants. China is the hometown of ginkgo, and the existing ginkgo resources account for more than 70% of the world's ginkgo resources. Ginkgo biloba is an excellent economic tree species integrating food, medicine, health care, cosmetics and materials [1] In recent years, ginkgo has also been widely used as an important greening tree species all over the country. Because Ginkgo biloba is a precious relict plant, it has many original characteristics. It is one of the plant varieties that scientists focus on and has been valued and studied by different scientific researchers....

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Application Information

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IPC IPC(8): C07K1/14
Inventor 何梅施大伟谢寅峰魏晓东
Owner NANJING FORESTRY UNIV
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