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Method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm

A gymnosperm, two-dimensional electrophoresis technology, which is applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problems of research lag and other problems, and achieve the effect of rapid method, prevention of protein degradation, and great practical application value.

Inactive Publication Date: 2008-11-26
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no effective method and technology for preparing gymnosperm pollen tube whole protein samples for two-dimensional electrophoresis, which leads to a lag in proteome research related to gymnosperms compared to angiosperms

Method used

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  • Method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm
  • Method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Preparation of white stem pollen tube whole protein two-dimensional electrophoresis sample solution and carrying out two-dimensional electrophoresis analysis

[0026] 1) Filtrate and collect the pollen tubes obtained by culturing the pollen grains of white stem pollen grains for 22 hours with liquid medium (0.0001g / mL boric acid, 0.0003g / mL calcium nitrate, 0.12g / mL sucrose), and quickly grind them into powder with liquid nitrogen;

[0027] 2) Suspend the powder obtained in step 1) in an acetone solution containing 0.125 g / ml trichloroacetic acid and 0.07% mercaptoethanol, and place it at -20°C for 2 hours;

[0028] 3) Centrifuge at 15000rpm (19117g) at 4°C for 10min, discard the supernatant, resuspend the pellet in three times the volume of -20°C pre-cooled acetone solution containing 0.07% mercaptoethanol, and place at -20°C for 16 hours ;

[0029] 4) Centrifuge at 15000rpm (19117g) at 4°C for 10min, discard the supernatant, resuspend the pellet in a solut...

Embodiment 2

[0032] Example 2. Separation and preparation of the whole protein sample in the pollen tubes of gymnosperm pine pine and carry out two-dimensional electrophoresis analysis

[0033] 1) Filter and collect the pollen tubes obtained by culturing the pollen grains of Pinus chinensis for 22 hours with liquid medium (0.0001g / mL boric acid, 0.0003g / mL calcium nitrate, 0.12g / mL sucrose), and quickly grind them into powder with liquid nitrogen;

[0034] 2) Suspend the powder obtained in step 1) in an acetone solution containing 0.1 g / ml trichloroacetic acid and 0.05% mercaptoethanol, and place it at -10°C for 1 hour;

[0035] 3) Centrifuge at 15000rpm (19117g) at 4°C for 10min, discard the supernatant, resuspend the pellet in three times the volume of -20°C pre-cooled acetone solution containing 0.05% mercaptoethanol, and place at -10°C for 12 hours ;

[0036] 4) Centrifuge at 15000 rpm (19117g) at 4°C for 10 minutes, discard the supernatant, resuspend the pellet in a solution containing...

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Abstract

This invention discloses a method for preparing electrophoretic sample of gymnosperm pollen tube protein. The method comprises: (1) suspending gymnosperm pollen tube powder in trichloroacetic acid solution, placing at -20~-4 deg.C, centrifuging, collecting the precipitate, resuspending the precipitate in trichloroacetic acid solution, placing at -20 deg.C for 2-16 h, centrifuging, collecting the precipitate, washing with 80% acetone solution, and removing acetone to obtain crude extract of gymnosperm pollen tube protein; (2) dissolving in pyrolytic buffer solution, centrifuging, collecting to supernatant to obtain the electrophoretic sample of gymnosperm pollen tube protein. The electrophoretic sample of gymnosperm pollen tube protein can be used for reversible electrophoresis, and is rapid, simple and can provide high quality electrophoretic pictures.

Description

technical field [0001] The invention relates to a method for preparing gymnosperm pollen tube whole protein, in particular to a method for preparing gymnosperm pollen tube whole protein two-dimensional electrophoresis samples. Background technique [0002] As the carrier of the male reproductive unit in the fertilization process of flowering plants, pollen tube has typical polar growth characteristics, so it is an ideal model system for studying cell morphogenesis, vesicle and organelle movement, and signal transduction pathways. Proteome analysis is an important technical means to study the response of proteins in pollen tubes to cell changes and environmental stimuli, which is conducive to in-depth understanding of the sexual reproduction process of higher plants and the mechanism of cell polarity growth. [0003] Choosing the right sample preparation method is a critical step in determining the success or failure of proteomic research. Cells must be disrupted efficiently...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/26C07K2/00
Inventor 林金星陈彤吴小琴陈艳梅汪小雄
Owner INST OF BOTANY CHINESE ACAD OF SCI
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