Preparation and extraction of chorion proteins from Salmo salar embryos at the pigmented eye stage for electrophoresis with SDS-polyacrylamide gel

Abstract

The chorion fulfills important functions in fish embryos, including protecting the embryo during development. The characterization of the protein profile of this envelope could be used as a bioindicator in the evaluation of the quality of embryonic development. The object of this work was to validate a standardized protocol for protein extraction from chorion of Salmo salar embryos at 280 accumulated thermal units (ATU) by comparing and combining existing methods. The protocol consists of consecutive washing of the chorion samples followed by protein extraction with the solution that was named SDS solution (Tris–HCl 100 mM (pH 8), Urea 8 M, 1% SDS, β-mercaptoethanol 300 mM and EGTA 10 Mm, and 1% protease inhibitor cocktail) and mechanical methods. Protein extraction is enhanced by a working temperature of 75 °C and use of a disperser. The protein concentration was quantified by Bradford Assay. After extraction, the samples were diluted (dilution factor 10) before reading against the calibration curve. After gel electrophoresis with a load of 3 µg of protein, staining showed more than 4 bands, with molecular weights between 25 kDa and 180 kDa. • The protein profile of fish chorion was between 25 kDa and 180 kDa. • Solution containing 1% SDS allows a higher extraction of proteins from the chorion of Atlantic salmon embryos with 280 ATU. • Chorion protein identification is a valuable tool in determining gamete and embryo quality in fish.

Keywords

• Method of preparation and extraction of chorion proteins from embryos at 280 accumulated thermal units (ATU) of Atlantic salmon (Salmo salar) for protein characterization by SDS-PAGE electrophoresis