Method for accumulating cotton phosphorylated protein

Abstract

The invention relates to a method for accumulating cotton phosphorylated protein, belonging to the field of biotechnology. The method comprises the steps of extracting cotton total protein and utilizing the Al(OH)3 affinity chromatography to accumulate cotton phosphorylated protein. By using the method, 12mu g of phosphorylated protein can be accumulated from 1mg of cotton total protein so that the accumulation efficiency is more than 6 times of that of the imported Qiagen kit. The two-dimensional electrophoresis technology and the phosphorylated protein specific dye ProQ DiamondTM (Invitrogen) dyeing test prove that the phosphorylated protein accumulated by the method has no non-phosphorylated protein. The method can replace the expensive imported kit, be used to accumulate phosphorylated protein efficiently and specificly and be widely used in researches such as phosphorylated proteomics and cellular signal.

Description

1. Technical field [0001] The invention relates to a method for enriching cotton phosphorylated protein, belongs to the field of biotechnology, and relates to the separation and enrichment of cotton phosphorylated protein. 2. Background technology [0002] Protein separation and purification analysis is a research hotspot in the field of biological separation analysis in recent years. Protein post-translational modification is an important factor affecting protein structure and function, and plays an important role in the regulation of biochemical pathways. Among them, protein phosphorylation is The most common and important post-translational modification is a hot spot and difficulty in proteomics research. Protein phosphorylation and dephosphorylation regulate almost the entire process of human life activities, including cell proliferation, development and differentiation, nerve activity, muscle contraction, metabolism, tumorigenesis, etc. Protein phosphorylation-dephospho...

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Problems solved by technology

But using TiO 2 Chromatographic column enriches phosphorylated proteins, and non-specific enrichment of phosphorylated proteins rich in acidic amino acids occurs

However, the phosphorylated protein enrichment chromatography column kit (Qiagen) based on this technology is not only expensive, but also has extremely low enrichment efficiency when used for plant phosphorylated protein enrichment. Taking cotton as an example, every 2.5mg total Protein can only enrich 5 μg of phosphorylated protein (2 μg / mg), which is far from meeting the needs of the experiment

In 2005, Wolschin reported that Al(OH) 3 Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide / hydroxide affinity chromatography (MOAC)[J].Proteomics, 2005, 5(17):4389-4397.), but when this method is used to enrich cotton phosphorylated proteins, the enrichment efficiency is also very low (5μg / mg), and it is difficult to enrich enough phosphoric acid for precious samples that are difficult to sample protein for further study

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