A Method for Extracting Rice Leaf Plasma Membrane Phosphorylated Proteins Suitable for Two-dimensional Electrophoresis

A phosphorylated protein and phosphorylated protein technology, applied in the field of proteomics, can solve problems such as difficulty in directly determining protein molecular weight and isoelectric point, loss of hydrophobic protein in plasma membrane protein, low abundance of phosphorylated protein in plasma membrane, etc. Achieve the effect of less vertical trailing and horizontal stripes, good repeatability and low cost

Active Publication Date: 2022-03-04
SOUTH CHINA AGRI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the existing studies on the phosphorylated proteome of plant plasma membranes, most of the strategies adopted are: firstly isolate and purify plant plasma membranes, then enzymatically hydrolyze and enrich phosphorylated peptides of plasma membrane proteins, and then analyze by chromatography and mass spectrometry. Carry out protein phosphorylation modification analysis; its advantages are good peptide solubility and high enrichment specificity, but the disadvantage is that protein identification only relies on a single peptide to cause confusion, it is difficult to directly determine the molecular weight and isoelectric point of the protein, and non-specific adsorption Still exist
[0004] At present, there are no related reports on the rice plasma membrane phosphorylated proteome. The main reason is that the monocot plasma membrane is difficult to purify, and the abundance of plasma membrane phosphorylated proteins is very low, and the hydrophobicity of the plasma membrane protein itself easily leads to protein loss, etc.

Method used

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  • A Method for Extracting Rice Leaf Plasma Membrane Phosphorylated Proteins Suitable for Two-dimensional Electrophoresis
  • A Method for Extracting Rice Leaf Plasma Membrane Phosphorylated Proteins Suitable for Two-dimensional Electrophoresis
  • A Method for Extracting Rice Leaf Plasma Membrane Phosphorylated Proteins Suitable for Two-dimensional Electrophoresis

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Embodiment 1

[0035] Example 1 A method for extracting rice leaf plasma membrane phosphorylated proteins suitable for two-dimensional electrophoresis

[0036] 1. Rice planting and leaf collection

[0037] The rice was an indica rice line CO39. Paddy rice was sown in plastic pots filled with paddy field soil, at 24-26 °C, 14 h light (about 250 μmol m -2 ·s -1 ) in a growth chamber. Manage according to conventional methods, and sample when the fourth leaf is fully unfolded. The 3rd and 4th leaves were taken respectively, and the fresh weight of the leaves was quickly weighed. After quick freezing in liquid nitrogen, they were stored in a -70°C refrigerator for later use.

[0038] 2. Extraction of coarse plasma membrane from rice leaves

[0039] Weigh 80 g of rice leaves and grind to powder with liquid nitrogen. Add 260mL extraction buffer (containing 50mmol / LBTP-MES buffer, 250mmol / L sucrose, 2mmol / L EDTA, 10% v / v glycerol, 0.5% w / v BSA, 10mmol / L PMSF, 2mmol / L DTT, 0.6 %w / v PVP k-30, 4...

Embodiment 2

[0044] Example 2 Two-dimensional electrophoresis analysis and specificity analysis of rice leaf plasma membrane phosphorylated proteins

[0045] 1. Analysis of purification effect of rice leaf plasma membrane

[0046] The rice leaf plasma membrane was purified using 6.3% w / w PEG 3350 / Dextran T-500 two-phase partition system. The results showed that after 100 mg of crude plasma membrane protein was partitioned three times, the yield of the purified plasma membrane protein was About 2.71 to 3.21%.

[0047] Using different inner membrane marker enzymes (H + -ATPase)-specific inhibitors to treat crude plasma membranes and purify plasma membranes to evaluate the purity of plasma membranes. The specific steps are: add 5 μg plasma membrane protein to 0.5mL BTP-MES buffer (containing 30mmol / LBTP-MES pH 6.5, 5mmol / L MgSO 4 , 50mmol / L KCl, 0.02% (w / v) Brij 58, 5mmol / L Na 2-ATP), start the reaction, incubate at 30°C for 30min, add 2.5mL terminator [containing 0.5mmol / L PVP k30, 86mmo...

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Abstract

The invention belongs to the technical field of proteomics, specifically discloses for the first time a method for extracting rice leaf plasma membrane phosphorylated proteins suitable for two-dimensional electrophoresis, including extraction of rice leaf rough plasma membranes, purification of rice leaf plasma membranes, and rice leaf plasma membrane extraction methods. Enrichment of membrane phosphorylated proteins and other steps. The extraction method of the present invention is simple in operation, high in extraction efficiency and low in cost, the purity of the obtained rice leaf plasma membrane can reach more than 93%, and the yield of plasma membrane phosphorylated protein enriched from plasma membrane protein is 3.33-3.89% . This method is suitable for plant materials that are difficult to extract plasma membrane phosphorylated proteins and have many interfering impurities; at the same time, the obtained plasma membrane phosphorylated proteins have fewer impurities and interfering substances, which can meet the requirements of two-dimensional electrophoresis. Uniform distribution, less longitudinal tailing and horizontal stripes, and good repeatability; this method is suitable for the preparation and analysis of monocot plasma membrane phosphorylated proteomes.

Description

technical field [0001] The invention relates to the technical field of proteomics, in particular to a method for extracting rice leaf plasma membrane phosphorylated proteins suitable for two-dimensional electrophoresis. Background technique [0002] Protein phosphorylation refers to protein phosphorylation under the action of protein kinase and dephosphorylation under the action of phosphatase. As one of the most important forms of protein post-translational modification, protein phosphorylation regulates almost all life activities in cells, especially plays an extremely important role in regulating cell signal transduction. Phosphoproteomics refers to the use of proteomics analysis methods to observe the modification status and changes of phosphorylated proteins in cells as a whole, and then analyze the regulation and molecular mechanism of specific phosphorylation modifications on life processes; its content It mainly includes the enrichment, detection, identification and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04C07K14/415C07K1/30
CPCC12N5/04C07K14/415
Inventor 聂燕芳李云锋王振中叶智坚邹小桃
Owner SOUTH CHINA AGRI UNIV
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