Extracting method for phosphorylated protein suitable for two-dimensional electrophoresis of plasma membranes of rice leaves

A technology of phosphorylated protein and extraction method, applied in the field of extraction of plasma membrane phosphorylated protein from rice leaves, can solve the problem of difficulty in directly determining protein molecular weight and isoelectric point, loss of hydrophobic protein of plasma membrane protein, and abundance of plasma membrane phosphorylated protein. To avoid problems such as low degree of plasma membrane phosphorylated protein, less impurities and interfering substances in plasma membrane, good repeatability, less vertical tailing and less horizontal streaks

Active Publication Date: 2019-01-04
SOUTH CHINA AGRI UNIV
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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

Method used

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  • Extracting method for phosphorylated protein suitable for two-dimensional electrophoresis of plasma membranes of rice leaves
  • Extracting method for phosphorylated protein suitable for two-dimensional electrophoresis of plasma membranes of rice leaves
  • Extracting method for phosphorylated protein suitable for two-dimensional electrophoresis of plasma membranes of rice leaves

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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 proteomic technology, and particularly discloses an extracting method for phosphorylated protein suitable for two-dimensional electrophoresis of plasmamembranes of rice leaves for the first time. The extracting method comprises the steps of extraction for coarse plasma membranes of the rice leaves, purification of the plasma membranes of the rice leaves, enrichment of the phosphorylated protein of the plasma membranes of the rice leaves and the like. The extracting method is simple in operation, high in extracting efficiency and low in cost, thepurity of the plasma membranes of the rice leaves can reach 93% or above, and the yield of the enriched phosphorylated protein of the plasma membranes from protein of the plasma membranes is 3.33-3.89%. The extracting method is suitable for the plant materials with difficultly extracted phosphorylated protein of the plasma membranes and fewer interferents; the obtained phosphorylated protein of the plasma membranes contains fewer impurities and interferents and can meet the requirements of two-dimensional electrophoresis, the obtained two-dimensional electrophoretogram is clear in background,uniform in protein spot distribution and fewer in longitudinal trailing and horizontal transverse grain phenomenon and has good repeatability, and the extracting method is suitable for preparation and analysis of phosphorylated proteomes of the plasma membranes of monocotyledons.

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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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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