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Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.