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Acquisition method for extraction of total lily protein and two-directional electrophoresis differential protein map

A protein map and two-dimensional electrophoresis technology, applied in the acquisition of two-dimensional electrophoresis differential protein map and the extraction of total lily protein, can solve problems such as unreported, and achieve the effect of uniform point distribution, many protein points, and clear map background.

Active Publication Date: 2016-04-27
FLOWER RES INST OF YUNNAN ACAD OF AGRI SCI
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Problems solved by technology

In recent years, protein two-dimensional electrophoresis technology has been widely used in the research of rice, wheat, cotton, corn, soybean and other crops, but the research on lily protein two-dimensional electrophoresis technology has not been reported at home and abroad

Method used

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  • Acquisition method for extraction of total lily protein and two-directional electrophoresis differential protein map
  • Acquisition method for extraction of total lily protein and two-directional electrophoresis differential protein map
  • Acquisition method for extraction of total lily protein and two-directional electrophoresis differential protein map

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Embodiment 1

[0046] Embodiment 1 The inventive method

[0047] The extraction of lily total protein provided by the present invention and the acquisition method of differential protein map of two-dimensional electrophoresis comprise the following steps:

[0048] (1) Extraction of lily total protein:

[0049] ①Weigh 1g of fresh lily leaves, put them in a mortar pre-cooled at -20°C, add 10ml of liquid nitrogen, and quickly grind them to powder.

[0050] ② Measure 20ml of 10% trichloroacetic acid-acetone solution with 1% phenylmethylsulfonyl fluoride and 0.1% dithiothreitol, wash the mortar three times, and wash the powder in the mortar into a 50ml centrifuge tube Quickly store in a -20°C refrigerator overnight; take the overnight centrifuge tube and centrifuge at 20,000 rpm for 20 minutes; discard the supernatant.

[0051] The preparation method of the 10% trichloroacetic acid-acetone solution of adding 1% phenylmethylsulfonyl fluoride and 0.1% dithiothreitol is: 10g trichloroacetic acid i...

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Abstract

The invention discloses an acquisition method for extraction of total lily protein and a two-directional electrophoresis differential protein map. In the method, the rotating speed is set to be 20000 rpm in the extraction of total lily protein, and the total lily protein is precipitated and centrifuged at least three times repeatedly, so that the extracted total lily protein is relatively white in color and good in quality; the mass detection step of the protein is added, and the mass of the extracted protein is detected in a sodium dodecyl sulfate polyacrylamide gel manner, so that the problem that the subsequent first direction isoelectric focusing effect is poor is avoided. The focusing program in the isoelectric focusing effect is improved, the time of two desalting steps such as step S1 and step S2 in the focusing program is prolonged to be 2.5h, and the salt of the protein is well removed, so that the focusing program is well completed. The two-directional electrophoresis differential protein map of the total lily protein obtained by the method is clear in background, more in protein spots and uniform in protein spot distribution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting lily total protein and obtaining a two-dimensional electrophoresis differential protein map. Background technique [0002] Lily (Lilium spp.) is known as the "king of flower bulbs" and is one of the top five fresh cut flowers in the world. According to statistics, the global bulb production area in 2009 was 35,749 hectares, of which 23,561 hectares were planted in the Netherlands, accounting for 66%, 5,400 hectares were planted in the UK, accounting for 15%, and 4,680 hectares were planted in China, accounting for 13%. The global trade volume of lily bulbs has reached more than 2 billion US dollars, and the annual trade volume has reached more than 2.5 billion. According to statistics from the Ministry of Agriculture, the sales volume of fresh-cut lilies in my country in 2013 reached 4.295 billion yuan, ranking first in the sales volume of fresh-c...

Claims

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Application Information

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IPC IPC(8): G01N27/447
CPCG01N27/44778
Inventor 张艺萍王继华杨秀梅瞿素萍许凤王丽花苏艳张丽芳
Owner FLOWER RES INST OF YUNNAN ACAD OF AGRI SCI
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