Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting.

Analysis of proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting (western blotting) is a vital part of the molecular biologist's toolkit. This technique separates a complex protein mixture by molecular weight and then assays the presence of target proteins using specific antibodies. Immunoblotting has a variety of applications. Examples include use as a targeted approach to study protein-protein interactors or as a control to confirm expression or depletion of protein targets. However, the successful execution of immunoblotting requires complicated, multistep experiments. Protocols must be optimized for each organism, target protein, and application. Therefore, knowledge gaps exist for the use of immunoblots in many models, including the model frog Xenopus laevis. Due to their large size, abundant material for biochemical experiments, and facile handling, X. laevis oocytes and embryos have been vital for studying principles of translational control. However, this species lacks specific protocols for robust and routine immunoblotting. Here, we offer an in-depth protocol for western blotting optimized for samples from multiple Xenopus developmental stages. We then analyze translational regulators across development.