Click-iT (R) Plus OPP Alexa Fluor (R) Protein Synthesis Assay in Embryonic Cells

This protocol describes a method to assess relative changes in the level of global protein synthesis in the preimplantation embryo using the Click-iT (R) Plus OPP Protein Synthesis Assays. In this assay, O-propargylpuromycin (OPP), an analog of puromycin, is efficiently incorporated into the nascent polypeptide of newly translated proteins in embryonic cells. OPP is fluorescently labeled with a photostable Alexa FluorTM dye and detected with fluorescence microscopy. The intensity of the fluorescence is quantitatively analyzed. This is a fast, sensitive, and non-radioactive method for the detection of protein synthesis in early embryo development. It provides a tool for analyzing the temporal regulation of protein synthesis, as well as the effects of changes in the embryonic microenvironment, and pharmacological and genetic modulations of embryo development.