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Ammopiptanthus monogolicus protein bidirectional electrophoresis technique

A two-dimensional electrophoresis, protein technology, applied in the field of high stress resistance plant research, can solve problems such as failure to successfully prepare high purity protein

Inactive Publication Date: 2011-04-20
XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the fact that plant materials in extreme environments contain a variety of secondary metabolites (pigments, phenols, quinones, etc.) produced under extreme conditions, many interferences make two-dimensional electrophoresis, the core technology of proteome research, a challenge. The method cannot successfully prepare high-purity proteins. During the electrophoresis process, reasonable parameter settings can effectively remove salt, focus, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Using Eppendorf ultracentrifuge, two-dimensional electrophoresis PROTEAN II Xi / XL Cell (BIO-RAD, USA)

[0027] a. Collect the leaves of Ilex japonica from Turpan Desert Botanical Garden, ship them back in a liquid nitrogen tank, and store them in the laboratory below -80°C for later use;

[0028] b. Weigh 5g of complete leaf samples of Ilex chinensis, rinse the leaves twice with diethyl ether pre-cooled at -20°C, remove lipids and waxy impurities, evaporate to dry at low temperature, and pulverize the samples with liquid nitrogen in a masher;

[0029] c. Take 1 gram of sample and shake 3 times weakly alkaline 25mmol / L pH8.9 tris buffer solution, 1mmol / L protease inhibitor phenylmethylsulfonyl fluoride in a centrifuge tube, and set aside at 4°C. Place for 1 hour at 4°C, centrifuge at 20,000×g, float off the upper layer of lipids, and subpackage the clarified filtrate for later use. This step can fully extract the total protein of the leaves, and can effectively prevent t...

Embodiment 2

[0043] Using Eppendorf ultracentrifuge, two-dimensional electrophoresis PROTEAN II Xi / XL Cell (BIO-RAD, USA)

[0044] a. Collect the leaves of Ilex japonica, ship them back in a liquid nitrogen tank, and store them in the laboratory below -80°C for later use;

[0045] b. Quantitatively weigh the complete leaf sample of Ilex chinensis, rinse the leaf twice with ether at -20°C to remove lipids and waxy impurities, and crush the sample with liquid nitrogen in a masher;

[0046] c. Shake 3 times weakly alkaline 25mmol / L pH8.9 Tris buffer solution and 1mmol / L protease inhibitor phenylmethylsulfonyl fluoride in a centrifuge tube, and let stand at 4°C for 1 hour. Temperature 4°C, centrifuge at 20000×g, float off the upper layer of lipids, and clarify the filtrate for later use; this step can fully extract the total protein of leaves, and can effectively avoid protein degradation;

[0047] d. Add 1.2 times 9mol / L urea, 2mol / L thiourea, 4% (weight to volume ratio) 3-[3-(cholamidopropy...

Embodiment 3

[0058] Using Eppendorf ultracentrifuge, two-dimensional electrophoresis PROTEAN II Xi / XL Cell (BIO-RAD, USA)

[0059] a. Collect the leaves of Ilex japonica, ship them back in a liquid nitrogen tank, and store them in the laboratory below -80°C for later use;

[0060] b. Weigh 5g of complete leaf samples of Ilex chinensis, wash the leaves twice with diethyl ether pre-cooled at -20°C, remove lipids and waxy impurities, evaporate to dry at low temperature, and pulverize the samples with liquid nitrogen in a masher;

[0061] c. Take 1g sample and shake 3 times of weakly alkaline 25mmol / L pH8.9 tris buffer solution, 1mmol / L protease inhibitor phenylmethylsulfonyl fluoride in a centrifuge tube, and keep the temperature at 4°C. Place for 1 hour at 4°C, centrifuge at 20,000×g, float off the upper layer of lipids, and clarify the filtrate for later use; this step can fully extract the total protein of the leaves, and can effectively avoid protein degradation;

[0062] d. Add 1.5 time...

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Abstract

The invention relates to the protein two-dimensional electrophoresis technology of a Mongolian ammopiptanthus, which adopts a two-dimensional electrophoresis technology to prepare the protein of a plant living in extreme environment. The experiment carried out to the Xinjiang ammopiptanthus living in the extreme environment by the technical proposal achieves good effect. At present, repeated experiments certify that: the two-dimensional electrophoresis technology has complete prepared laboratory samples, good repeatability, clear atlas and reliable results, can provide technical examples for the research of plant proteomics threatened by adverse circumstances and is a two-dimensional electrophoresis technology applicable to the proteomic analysis of the Xinjiang ammopiptanthus.

Description

technical field [0001] The invention relates to a protein two-dimensional electrophoresis technology of holly holly, which is a proteomics study of holly holly in Xinjiang that survives in an extreme environment. The technology can be directly applied to the research of highly stress-resistant plants that survive in extreme conditions. Background technique [0002] Stress resistance is a countermeasure that plants acquire in order to adapt to adverse environments in the process of systematic evolution. In the living environment of plants, some abiotic factors stress will have a serious impact on the growth and survival of plants. In order to cope with the adverse effects of the external environment, plants have formed a complete defense mechanism during the evolution process. [0003] Sand holly (Ammopiptanthus Cheng f.) is the only endemic evergreen leguminous broad-leaved shrub group that survives in the Gobi Desert in central Asia in the Pan-Arctic region. Xinjiang holly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/28C07K1/26
Inventor 鲁春芳尹林克牟书勇尉姗姗赵峰侠
Owner XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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