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Method for preparing buffalo testis convoluted seminiferous tubule total protein samples and two-dimensional electrophoresis separation method

A technology of seminiferous tubules and separation method, which is applied in the field of preparation of total protein samples of buffalo testicular seminiferous tubules, which can solve the problems of discrimination, human error, limited range of protein separation, and affecting the effectiveness and reliability of two-dimensional electrophoresis technology

Active Publication Date: 2014-07-30
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technology itself has shortcomings such as limited protein separation range, discrimination effect and large human error, especially the preparation of the analyzed protein samples greatly affects the effectiveness and reliability of the two-dimensional electrophoresis technology.

Method used

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  • Method for preparing buffalo testis convoluted seminiferous tubule total protein samples and two-dimensional electrophoresis separation method
  • Method for preparing buffalo testis convoluted seminiferous tubule total protein samples and two-dimensional electrophoresis separation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Collection and purification of fine tubes of buffalo testis

[0029] (1) Obtain juvenile buffalo testes from local slaughterhouses (disinfect buffalo scrotum skin with 75% alcohol by volume, incise the scrotum and peel off its surrounding tissues, and take out the testes), store them in 37°C normal saline and bring them back to the experiment quickly chamber; wash with PBS repeatedly for 3 times, remove the capsule and albuginea, cut out a small piece of testicular parenchyma, tear the tissue slowly with tweezers and place it in a 15mL glass tube;

[0030] (2) Add the mixed enzyme solution of collagenase IV and DNase I to the 15mL glass tube in step (1) (dissolve collagenase IV and DNase I in PBS to make solutions with concentrations of 1mg / mL and 300μg / mL respectively, and then Mix according to the volume ratio of 10:3 to obtain), so that the mass volume ratio of testicular tissue and mixed enzyme solution is 1:10, place on a shaker at 37°C and shake for 2 to 3 hour...

Embodiment 2

[0042] 1. Collection and purification of fine tubes of buffalo testis

[0043] (1) Obtain aged buffalo testes from local slaughterhouses (disinfect buffalo scrotum skin with 75% alcohol by volume, incise the scrotum and peel off its surrounding tissues, and take out the testes), store them in 37°C normal saline and bring them back to the experiment quickly room. Rinse with PBS repeatedly for 3 times, remove the capsule and albuginea, cut a small piece of testicular parenchyma, tear the tissue slowly with forceps and place it in a 15mL glass tube;

[0044] (2) Add the mixed enzyme solution of collagenase IV and DNase I to the 15mL glass tube in step (1) (dissolve collagenase IV and DNase I in PBS to make solutions with concentrations of 1mg / mL and 300μg / mL respectively, and then Mix according to the volume ratio of 10:3 to obtain), so that the mass volume ratio of testicular tissue and mixed enzyme solution is 1:10, place on a shaker at 37°C and shake for 2 to 3 hours, until a...

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Abstract

The invention discloses a method for preparing buffalo testis convoluted seminiferous tubule total protein samples. The method belongs into a lysate extraction-acetone precipitation method and is simple and feasible, fast and reliable and is used in combination with combinative enzymes to effectively digest and remove testis muscle tissue, connective tissue and other tissue components and DNA, and ultimately a plenty of white testis convoluted seminiferous tubules are enriched; the obtained convoluted seminiferous tubules are relatively pure and free of other impurities, the extracted total protein has relatively high quality. The method is suitable for two-dimensional electrophoresis analysis. Accordingly, the inventors establish a set of two-dimensional electrophoresis separation method for buffalo testis convoluted seminiferous tubule total protein samples by trial and error and optimization of a two-dimensional electrophoresis technology system. The electrophoretogram with relatively high quality and relatively high resolution, which can be obtained by the method, offers very important applicable values to the subsequent mass spectrometry and the protein identification.

Description

technical field [0001] The invention belongs to the technical field of protein extraction and proteomics separation, and in particular relates to a preparation method of a buffalo testicular seminiferous tubule total protein sample and a two-dimensional electrophoresis separation method. Background technique [0002] The seminiferous tubules in the testis are important places for spermatogenesis. The seminiferous tubules contain spermatogonia and supporting cells. The spermatogonia differentiate into germ cells at all levels through their own division and proliferation and under the secretion of supporting cells. Sertoli cells play a role in supporting, nourishing and protecting spermatogenic cells, and play a vital role in the final development of spermatocytes into sperm with a complete shape. At present, most of the domestic and foreign studies on the spermatogonia or Sertoli cells are isolated and cultured in vitro, and few involve the extraction of the total protein of ...

Claims

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Application Information

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IPC IPC(8): C07K1/30G01N1/28G01N27/26
CPCY02P20/141
Inventor 张明黄愉淋付强黄德伦
Owner GUANGXI UNIV
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