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.