Characterization of concanavalin A precipitated proteins from early mouse embryos: a 2-dimensional gel electrophoresis study

Concanavalin A (Con A), a plant lectin which binds to mannose-containing components, has been used to precipitate a specific class of proteins synthesized during preimplantation mouse embryogenesis. Total cellular protein was extracted from labeled embryos with Nonidet P40, reacted with Con A, and then exposed to rabbit antiserum directed against Con A. Immune complexes were precipitated with protein A from Staphylococcus aureus. After washing, Con A-bound proteins were eluted with mannoside which released between 3 and 10% of the total radioactivity from unfertilized and fertilized eggs, 2-cell and 8-to 16-cell embryos, and late blastocysts. In control experiments, in which mannoside was added prior to the Con A, only 0.1–0.5% of the total radioactivity was eluted, indicating little release of proteins nonspecifically bound to the protein A-anti-Con A-Con A complex. When these precipitates were examined by two-dimensional gel electrophoresis, approximately 30–70 peptides in the defined periods of synthesis were detected in autoradiograms. To precipitate cell surface proteins, intact embryos were incubated in Con A and anti-Con A prior to extraction with Nonidet P40. Less than 1.0% of the total radioactivity was eluted with mannoside. A complex of three pairs of peptides was present in 2-cell and 8- to 16-cell embryos. The qualitative changes in two-dimensional gel patterns of Con A-binding proteins has provided a program of synthesis occurring during normal development for a specific class of proteins.