Method for accumulating cotton phosphorylated protein

A phosphorylated protein and cotton technology, applied in the biological field, can solve the problems of high price, non-specific enrichment of phosphorylated protein, and low enrichment efficiency, and achieve the effect of low price

Inactive Publication Date: 2011-02-09
NANJING AGRICULTURAL UNIVERSITY
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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), wh...

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  • Method for accumulating cotton phosphorylated protein
  • Method for accumulating cotton phosphorylated protein
  • Method for accumulating cotton phosphorylated protein

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Embodiment Construction

[0032] (1) Determination of the best cotton total protein extraction method

[0033] 1. First, the total protein of cotton was extracted by phenol method and TCA+phenol method respectively.

[0034] The present invention uses cotton ovules or leaves as experimental materials to extract the total protein of cotton, and successively explores two methods for extracting the total protein—the phenol method and the TCA+phenol method.

[0035] (1) Phenol method: Add the same amount of PVPP and quartz sand to 1 g of cotton ovule or leaf, grind it to powder in liquid nitrogen, add 5 mL of extraction buffer, 15 mL of pH8.0 Tris-HCl saturated phenol, vortex for 10 min, at 4 °C Let stand for 30min. Centrifuge at 10,000 g for 10 min at 4°C. Collect the phenol phase, add 5 times the volume of ice acetone, centrifuge at 15,000 g for 15 min at -20°C overnight, suspend the precipitate in cold methanol, wash twice, and dry in vacuum at 4°C to obtain the total protein, which is sealed and stored...

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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...

Claims

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Application Information

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IPC IPC(8): C07K1/22
Inventor 刘康郝佩佩
Owner NANJING AGRICULTURAL UNIVERSITY
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