Method for detecting gymnosperm pollen in-tube antigen

A technology for gymnosperms and pollen tubes is applied in the field of detecting antigens in gymnosperms pollen tubes by using fluorescent immunological technology, which can solve the problems of difficult antigen detection and positioning, antigen inactivation, and lag in biological research, and achieves prevention of rapid quenching of fluorescence, convenient and convenient Operation, marking effect is good

Inactive Publication Date: 2006-08-02
INST OF BOTANY CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In addition, for some protein antigens, due to the easy inactivation of the antigen during the fixation process and the limitation of the time and temperature conditions required for the antigen-antibody reaction process, it is difficult to detect and locate the antigen in the pollen tube, and there is no related gymnosperm so far. A proven method for the detection and localization of antigens in pollen tubes has led to a lag in biological research related to gymnosperms relative to angiosperms

Method used

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  • Method for detecting gymnosperm pollen in-tube antigen
  • Method for detecting gymnosperm pollen in-tube antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, the detection of the antigen in the pollen tube of green skewer

[0026] Take the green skewer pollen tubes that have been cultivated for 18 hours, suspend them with sterile water to make a pollen tube suspension, and perform the following steps:

[0027] 1) Collect pollen tubes, resuspend with pH7.2 PBS buffer (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride), and repeat this step once;

[0028] 2) Add paraformaldehyde to make the final concentration 2% (freshly prepared), incubate at room temperature for 2 hours, absorb the paraformaldehyde, and use PBS buffer (10mM phosphate, 138mM sodium chloride, 2.7mM chloride Potassium chloride) fully washed twice, 5min each time;

[0029] 3) Enzymolysis solution (pH5.5, 15mMMes , 400mM mannitol, 5mM calcium chloride and 1μg·mL -1 Protease inhibitors (aprotinin, Calbiochem) enzymatically hydrolyze the pollen tubes of green skewers for 10 minutes, and the pollen tubes after enzymolysis are immediat...

Embodiment 2

[0033] Example 2, the detection of antigens in the pollen tubes of Pine pine

[0034] Take the white-bark pine pollen tubes cultivated for 72 hours, suspend them with sterile water to make a pollen tube suspension, and carry out the following steps:

[0035] 1) Collect pollen tubes, resuspend with pH7.2 PBS buffer (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride), and repeat this step once;

[0036] 2) Add paraformaldehyde to make the final concentration 4% (freshly prepared), incubate at room temperature for 1 hour, absorb the paraformaldehyde, and wash with PBS buffer solution (10mM phosphate, 138mM sodium chloride, 2.7mM chloride) with pH 7.2 Potassium chloride) fully washed twice, 5min each time;

[0037]3) Enzyme hydrolyzate (pH5.5, 15mM) containing 3% cellulase R-10 (purchased from Yakult Honsha Co.Ltd) and 2% macerozyme R-10 (purchased from Yakult Honsha Co.Ltd) Mes, 400 mM mannitol, 5 mM calcium chloride and 1 μg mL -1 Protease inhibitors (pepstatin,...

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Abstract

The present invention discloses a method for detecting gymnosperm pollen tube interior antigen. Said method includes the following steps: (1), using phosphoric acid buffer solution containing paraformaldehyde to fix pollen tube, using zymolysis solution containing cellulose and macerozyme to zymolyze gymnosperm pollen tube, using Triton X-100 or NP-40 aqueous solution whose mass percentage concentration is 0.2-1% to wash the pollen tube; and (2), making the pollen tube treated by step 1 and the correspondent antibody for resisting the antigen to be tested undergo the process of immunofluorescence reaction, if the fluorescence is detected, the gymnosperm pollen tube interior contains antigen to be tested and can obtain the location condition of said antigen to be tested.

Description

technical field [0001] The invention relates to a method for detecting antigens in gymnosperm pollen tubes, in particular to a method for detecting antigens in gymnosperm pollen tubes by means of fluorescence immunity technology. Background technique [0002] As the carrier of the male reproductive unit in the fertilization process of flowering plants, the pollen tube carries and transmits the sperm cells to the ovule, realizes the combination of sperm and eggs, and completes fertilization. Pollen tube is a typical haploid cell with apical growth characteristics and independent existence. It shows a uniform columnar polar growth pattern when grown in vitro, so it is considered to be an ideal cell system for studying polar growth in the field of biotechnology. In addition, as a model system, pollen tube is also of great significance for in-depth study of cell polar growth, cell-cell interaction and signal transduction. Since the pollen tube cell wall is mainly composed of ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/543G01N21/76
Inventor 林金星张凌云
Owner INST OF BOTANY CHINESE ACAD OF SCI
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