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Method for acquiring two-dimensional electrophoretogram of nectar protein of liriodendron tulipifera by aid of two-dimensional electrophoresis system

A technology of two-dimensional electrophoresis and electrophoresis, which is applied in the field of plant proteomics, can solve the problem of high-quality two-dimensional electrophoresis of samples without effective proteomics analysis, and achieve high protein extraction efficiency, optimized extraction process, and high resolution. Effect

Inactive Publication Date: 2016-12-07
NANJING FORESTRY UNIV
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  • Description
  • Claims
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Problems solved by technology

[0006] Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the invention is to provide a method for obtaining the two-dimensional electrophoresis spectrum of the nectar protein of Liriodendron tulipifera by using the two-dimensional electrophoresis system, so as to solve the problem that there is no effective and applicable method in the current domestic prior art. Preparation of samples for proteomic analysis of Liriodendron tulipifolia nectar and problems in obtaining high-quality 2D electrophoresis patterns

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  • Method for acquiring two-dimensional electrophoretogram of nectar protein of liriodendron tulipifera by aid of two-dimensional electrophoresis system
  • Method for acquiring two-dimensional electrophoretogram of nectar protein of liriodendron tulipifera by aid of two-dimensional electrophoresis system
  • Method for acquiring two-dimensional electrophoretogram of nectar protein of liriodendron tulipifera by aid of two-dimensional electrophoresis system

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Embodiment 1

[0047] A method for obtaining the two-dimensional electrophoresis spectrum of the nectar protein of Liriodendron tulipifera by using a two-dimensional electrophoresis system, comprising the steps of:

[0048] (1) Preparation of protein samples from Liriodendron tulipifera nectar

[0049] During the period when Liriodendron tulipifolia secretes a lot of nectar and the flower buds are not open, carefully peel off the tepals, such as figure 1 As shown, the nectar was aspirated with a micropipette, then snap-frozen in liquid nitrogen and brought back. During the suction process, avoid the pipette tip from piercing the flower organ tissue, causing tissue fluid to contaminate the nectar.

[0050] Take 10 mL of Liriodendron tulipifera nectar stored at -80°C, put it on ice to melt, first filter the sample with an ultra-microfiltration membrane with a pore size of 0.22 μm, and then enrich and purify the protein in each Ultra-0.5 Centrifugal Filter 3K Add 500μL sample to the column, c...

Embodiment 2

[0066] The method for obtaining the two-dimensional electrophoresis spectrum of the nectar of Liriodendron tulipifera nectar using conventional protein extraction methods comprises the following steps:

[0067] (1) Preparation of protein samples from Liriodendron tulipifera nectar

[0068] During the period when Liriodendron tulipifolia secretes a lot of nectar and the flower buds are not open, carefully peel off the tepals, such as figure 1 As shown, the nectar was aspirated with a micropipette, then snap-frozen in liquid nitrogen and brought back. During the suction process, avoid the pipette tip from piercing the flower organ tissue, causing tissue fluid to contaminate the nectar.

[0069] Liriodendron tulipifera nectar proteins were extracted using the most commonly used conventional TCA-acetone method. Take 10 mL of Liriodendron tulipifera nectar stored at -80 °C, put it on ice to melt, add 3 times the volume of -20 °C pre-cooled 10% TCA solution (w / v) prepared in aceto...

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Abstract

The invention discloses a method for acquiring a two-dimensional electrophoretogram of nectar protein of liriodendron tulipifera by the aid of a two-dimensional electrophoresis system. After a nectar sample is filtered by an ultramicro filter membrane, the nectar protein is concentrated and purified by a protein ultrafiltration and concentration purification column, the concentration of the purified and concentrated nectar protein is detected with a Bradford method, the molecular weight distribution range of the nectar protein of the liriodendron tulipifera is pre-detected through one-dimensional lauryl sodium sulfate polyacrylamide gel electrophoresis, finally, proper standard protein and a gel strip with a proper length and pH range are selected according to a one-dimensional electrophoresis result, the nectar protein of the liriodendron tulipifera is separated with the two-dimensional electrophoresis system, and the two-dimensional electrophoretogram is acquired. The method has the characteristics that the operation is simple, the protein extraction rate is high and few interfering substances exist and is suitable for special materials such as liquid samples, the high-quality two-dimensional gel electrophoretogram with high resolution ratio, clear protein points, larger numbers, uniform distribution and clear background can be obtained, and the experiment repeatability and the stability are good.

Description

technical field [0001] The invention belongs to the technical field of plant proteomics, and in particular relates to a stable and efficient method for obtaining a two-dimensional electrophoresis spectrum of the nectar protein of Liriodendron tulipifera by using two-dimensional electrophoresis technology. Background technique [0002] Proteome refers to "all proteins expressed by genes in cells, tissues or organisms under specific physiological conditions". Proteomics is a systematic study of all proteins in an organism, including protein changes, translation Post-modification and protein-protein interactions. Proteomics research can have a more comprehensive understanding of various physiological processes of organisms at the protein level, and has become a hot spot in life science research. [0003] Two-dimensional electrophoresis (2-DE) is one of the commonly used protein separation techniques in proteomics research. The first dimension uses isoelectric point (pI) focus...

Claims

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Application Information

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IPC IPC(8): G01N27/447G01N1/34
CPCG01N27/44778G01N1/34
Inventor 陈金慧周艳威施季森杨立明查红光郑晨龙晓飞盛宇
Owner NANJING FORESTRY UNIV
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