Studies On Extraction Of Cattle Sperm Plasma Membrane And Separation Of Its Total Protein By Two-dimensional Gel Electrophoresis

In order to search differential proteins of X and Y sperm and separate X and Y sperm, the method of extraction of plasma membrane(PM) and the technique of two-dimensional gel electrophoresis in separating the protein of cattle spermatozoa were studied. We chose the method of glass grinding and Parr cell disruption bomb.PM was isolated by ultra centrifugation and differential sucrose dense centrifugation. A trace of PM was observed by enlarge 3000 with Scanning Electron Micrograph.The protein of cattle spermatozoa was separated by two-dimensional gel electrophoresis technique, the effects of various sample preparation methods, loading quantities and isoelectric focusing electrophoresis were studied .The satisfied result was obtained when two step method of urea-guanidine hydrochloride lysed sperm and 13 cm IPG strip was used to conduct isoelectric focusing electrophoresis. More than 800 protein spots were detected by the imaging analysis system.The molecular weight and isoelectric point of most protein spots were 10 to 100 KD and 4.0 to 9.0 respectively. The protein map was set up.Two protein spots were selected and conducted with MALDI-TOF MS analysis after in-gel enzyme digestion. Spot Picker 2 got satisfied peptide mass finger-printing(PMF).These peptides were identified after searching database on the internet. Among these , seven were matched peptide, coverage sequence was 32%. The results met standard that matched peptides ranged from 6 to 20 and coverage sequence was over 15%.The study provided a basis for detecting and analyzing the differential proteins between X and Y sperm .