Method for extracting and separating total proteins of mice sperms by using two-dimensional electrophoresis

Abstract

The invention discloses a method for extracting and separating total proteins of mice sperms by using two-dimensional electrophoresis. The method comprises the following steps: preparing sperms, precipitating DNAs of the sperms after lysis of the sperms, preparing sperm proteins, preparing a protein extraction sample, carrying out first-dimensional isoelectric focusing electrophoresis, carrying out second-dimensional SDS-polyacrylamide gel electrophoresis, dyeing by using coomassie brilliant blue, preparing gel and scanning by using an image scanner. According to the method, lysis of the sperms is completely performed by Trizol, the proteins are extracted by chloroform, impurities are removed by isopropanol, guanidine hydrochloride and ethyl alcohol, and the proteins are purified by acetone, so that the problems of low content of the mice sperms, collection difficulty, small cell volume, a small amount of cytoplasm and a large amount of acrosomal protease are well solved; the sample prepared by the method is complete to prepare, has a large quantity of protein isolating points, and is high in repeatability and clear in chromatogram; by virtue of detection of PDQuestsoftware, the number of the protein points is greater than 1,000; therefore, the method lays the foundation for further mass spectrometry.

Description

technical field [0001] The invention belongs to the technical field of reproductive biology and disease pathogenesis research, and specifically relates to a method for extracting total protein of mouse sperm and two-dimensional electrophoresis. Background technique [0002] With the development of modern industry, the toxic and harmful substances in the environment have increased. In recent years, the influence of toxic and harmful substances on the reproductive system and the development of drugs have become research hotspots. Sperm is a highly differentiated cell, the mature reproductive cell of male animals. The main function of sperm is to complete the fertilization process and to continue the population. The abnormal structure and function of sperm can not only affect the quality of semen, but also one of the important reasons for male infertility. Gene is the genetic material of an animal, and its content is relatively constant; protein is the executor of gene funct...

AI Technical Summary

Problems solved by technology

The preparation of protein samples and the exploration of electrophoresis conditions are the key steps of two-dimensional electrophoresis. However, due to the small amount of sperm in mice, it is difficult to collect, the cells are small, the cytoplasm is less, and they are rich in acrosome protease. The preparation of protein samples has always been difficult.

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