Electrophoretic and zymographic characterization of proteins isolated by various extraction methods from ejaculated and capacitated boar sperms
Abstrakt
The presented work focuses on electrophoretic and zymographic characterization of boar sperm proteins isolated by various extraction methods and on comparison of the protein profiles obtained from ejaculated and in vitro capacitated spermatozoa. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated with the following agents: 1% v/v Triton X-100, 1% v/v Triton X-114, 2% v/v acetic acid, 1% m/v sodium dodecyl sulphate (SDS), 30mM N-octyl-beta-D-glucopyranoside (OBG), rehydration buffer (RHB) for isoelectric focusing and finally by the freezing-thawing approach.
The extracts were characterized in terms of 1-DE, 2-DE protein profiles, 1-DE glycoprotein staining and proteinase and hyaluronidase substrate zymographic profiles. The results have shown quantitative and qualitative differences in 1-DE protein and glycoprotein profiles with respect to the employed isolation approach.
These differences were seen even more clearly in 2-DE protein profiles, where it was possible to distinguish the presence/absence, changes in relative abundance and pI/M(r) shifts of various protein spots. Proteinase and hyaluronidase zymograms supported the prediction that various isolation protocols result in various profiles of enzymatically active molecules.