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

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...

AI Technical Summary

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.

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