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Method for extracting and detecting fish sperm membrane protein

A fish sperm and membrane protein technology, applied in peptide preparation methods, chemical instruments and methods, measuring devices, etc., to achieve the effects of short time-consuming, high yield and purity, efficient and rapid extraction

Inactive Publication Date: 2014-04-02
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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However, there are no reports on fish sperm membrane proteins

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  • Method for extracting and detecting fish sperm membrane protein
  • Method for extracting and detecting fish sperm membrane protein

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Embodiment 1

[0021] In this embodiment, the Yangtze River cuttlefish was used as the test object, and the sperm protein was extracted and detected.

[0022] The reagents used in this embodiment and the preparation method are as follows:

[0023] (1) Liquid A: 0.25M sucrose, 0.1M Tris-HCl, 0.5M benzamide, 1mM EDTA, mixed in equal volumes to obtain Liquid A;

[0024] Liquid B: the mass ratio is 5% fructose, 0.01M sodium citrate, mixed in equal volumes to obtain liquid B;

[0025] (2) Suspension buffer: 0.01M PBS buffer plus 0.05% streptavidin conventional solution preparation method, mixed with equal volumes, used for subsequent suspension treatment of sperm extracts, suspension centrifugation to collect supernatant;

[0026] (3) Sperm membrane protein separation solution: 1% Triton X-100, 1.7% Triton X-114, 1M NaCl solution, mixed in equal volumes to prepare a sperm membrane protein separation solution, which is used to extract fish sperm membrane protein;

[0027] (4) 5×SDS-PAGE sample s...

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Abstract

The invention discloses a method for extracting and detecting fish sperm membrane protein. The method comprises the following steps: pretreatment of a sample, fabrication of sperm suspension, suspension treatment of sperms, sperm membrane protein separation, sperm membrane protein degreasing purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection of sperm membrane protein and mass spectrometric detection of the sperm membrane protein. By adopting the method, the characteristics of a protease inhibitor and a TM-PEK (transmembrane-protein extraction kit) method are fitted on the basis of the traditional decontamination method, the cover degree of the extracted fish sperm membrane protein can be up to over 86%, and 0.6mg of membrane protein and 0.8mg of cytoplasmic protein can be obtained from 5*10<7> sperms on average. The basic data can be provided for verification of a fish sperm egg mechanism and a fertilization mechanism by the popularization and application of the method disclosed by the invention, guidance can be provided for culture and proliferation of fishes, especially reproductive breeding of migratory fishes in estuary sea, and a useful reference method is provided for research of sperm membrane protein of other aquatic species.

Description

technical field [0001] The invention belongs to the field of animal sperm detection, in particular to a method for extracting and detecting fish sperm membrane protein. Background technique [0002] Animal sperm is a type of cell with very specialized structures and functions. There are many membrane proteins and protein receptor complexes on the surface of sperm membranes, which are closely related to reproductive organisms such as spermatogenesis, maturation and capacitation, sperm egg recognition, and fertilization. Learning activities have important functions. [0003] Sperm membrane surface protein is an important molecule in spermatogenesis, maturation and sperm-egg interaction, and its in-depth study will help reveal the biological mechanism of spermatogenesis, maturation capacitation and fertilization. The surface of the plasma membrane of mature sperm contains a variety of proteins, which are inherent in themselves or adhere to the surface of the sperm, and play an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14G01N27/62
Inventor 方弟安张敏莹徐东坡刘凯周彦锋段金荣
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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