Extraction and dimensional electrophoresis method of mangrove plant total protein

A mangrove plant and two-dimensional electrophoresis technology, which is applied in the field of proteomics of mangrove plants, can solve the problems of difficult protein extraction and few application reports, and achieve the effect of clear background, good separation and reduced impurities in the electrophoretic spectrum

Inactive Publication Date: 2012-01-18
ZHEJIANG MARICULTURE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, protein extraction from mangrove plants is more difficult than other plants, with its particularity and complexity
However, the application of two-dimensional electrophoresis technology in plants is mainly concentrated on rice, wheat or some other herbaceous plants, and there are fewer reports on the application of mangrove plants.

Method used

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  • Extraction and dimensional electrophoresis method of mangrove plant total protein
  • Extraction and dimensional electrophoresis method of mangrove plant total protein
  • Extraction and dimensional electrophoresis method of mangrove plant total protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Using Eppendorf ultracentrifuge, two-dimensional electrophoresis PROTEANIIXi / XL Cell (BIO-RAD, USA)

[0028] (1) Collect fresh leaves of candela (two pairs of tender leaves);

[0029] (2) Weigh 1g of mesophyll, put it in liquid nitrogen and grind it thoroughly; transfer the powder to a centrifuge tube, add 4 times the volume of 20% TCA-acetone solution, vortex, and settle overnight at 4°C;

[0030](3) Collect the precipitate after centrifugation at 12000r / min at 4°C for 30min, wash the precipitate with pre-cooled acetone (containing 2% β-mercaptoethanol) at 4°C, vortex, and centrifuge at 12000r / min at 4°C for 10min to collect the precipitate , repeated three times, and the precipitate was dried at room temperature;

[0031] (4) Add lysis buffer (2mol / L thiourea, 7mol / L urea, 4% CHAPS, 40mmol / L Tris, 2mmol / L TBP to the crude protein extract of mangrove plant candela leaves obtained in step (3). , 0.2% Bio-lyte), dissolved overnight at 4°C, centrifuged at 12,000r / min fo...

Embodiment 2

[0046] Using Eppendorf ultracentrifuge, two-dimensional electrophoresis PROTEANIIXi / XL Cell (BIO-RAD, USA)

[0047] (1) Collect fresh leaves of candela (two pairs of tender leaves);

[0048] (2) Weigh 1g of mesophyll, put it in liquid nitrogen and grind it thoroughly; transfer the powder to a centrifuge tube, add 4 times the volume of 20% TCA-acetone solution, vortex, and settle overnight at 4°C;

[0049] (3) Collect the precipitate after centrifugation at 12000r / min at 4°C for 30min, wash the precipitate with pre-cooled acetone (containing 2% β-mercaptoethanol) at 4°C, vortex, and centrifuge at 12000r / min at 4°C for 10min to collect the precipitate , repeated three times, and the precipitate was dried at room temperature;

[0050] (4) Add lysis buffer (2mol / L thiourea, 7mol / L urea, 4% CHAPS, 40mmol / L Tris, 2mmol / L TBP to the crude protein extract of mangrove plant candela leaves obtained in step (3). , 0.2% Bio-lyte), dissolved overnight at 4°C, centrifuged at 12,000r / min f...

Embodiment 3

[0066] Using Eppendorf ultracentrifuge, two-dimensional electrophoresis PROTEANIIXi / XL Cell (BIO-RAD, USA)

[0067] (1) Collect fresh leaves of the mangrove plant tung flower (two pairs of young leaves);

[0068] (2) Weigh 1g of mesophyll, put it in liquid nitrogen and grind it thoroughly; transfer the powder to a centrifuge tube, add 4 times the volume of 20% TCA-acetone solution, vortex, and settle overnight at 4°C;

[0069] (3) Collect the precipitate after centrifugation at 12000r / min at 4°C for 30min, wash the precipitate with pre-cooled acetone (containing 2% β-mercaptoethanol) at 4°C, vortex, and centrifuge at 12000r / min at 4°C for 10min to collect the precipitate , repeated three times, and the precipitate was dried at room temperature;

[0070] (4) Add lysis buffer (2mol / L thiourea, 7mol / L urea, 4% CHAPS, 40mmol / L Tris, 2mmol / L TBP, 0.2% Bio-lyte), dissolved overnight at 4°C, centrifuged at 12,000r / min for 30min at 4°C, and the supernatant was the Tung flower leaf p...

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PUM

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Abstract

The invention provides an economical, simple, easily operated and rapid extraction and dimensional electrophoresis method of mangrove plant total protein. Repeated experiments have proved that a sample prepared by the method has good repeatability and an intelligible spectrum, and the technical scheme is especially suitable for an extraction and dimensional electrophoresis method of mangrove plant total protein. The invention employs 20% TCA-acetone solution to extract protein; 20% TCA can better remove salt ion, and acetone with a concentration of 80% has substantial effect on separating of a protein and condensed tannin compound, and can better separate the compound, remove tannin and increase protein concentration; the mangrove plant protein extracted by 20% TCA-acetone has high content and an intelligible 2-DE electrophoresis pattern; besides, the method is simply operated, with good repeatability and low reagent toxicity.

Description

technical field [0001] The invention relates to the proteomics of mangrove plants, in particular to the extraction of its total protein and the method of two-dimensional electrophoresis. Background technique [0002] Mangrove is a rare woody viviparous plant. It grows in tidal flats and shoals at the junction of tropical and subtropical land and sea, and is a special ecosystem where land transitions to sea. Mangroves shoulder the dual mission of optimizing the environment and promoting economic and social development, and have unparalleled ecological value. One of the most important ecological security guarantee systems for ecological balance in the southern coastal areas of China. In particular, the ecological, economic, social, cultural, regeneration functions and other values ​​generated by the mangrove ecosystem have been widely valued in China and the world. As the global climate warms, mangroves move northward in response to climate change. With the advancement of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/30C07K1/14C07K1/28C07K1/26
Inventor 郑春芳于冯陈少波施梦茄刘伟成艾为明谢起浪李尚鲁陈骁仇建标周志明黄丽马小伟
Owner ZHEJIANG MARICULTURE RES INST
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