Analysis of Phosphorylation Status of Ectopically Expressed Proteins in Early Xenopus Embryos

Xenopus embryos provide a rapid and accessible in vivo model, expressing a plethora of endogenous kinase and phosphatase enzymes that control protein phosphorylation and, in turn, affect physiological function. Traditionally, the detection of protein phosphorylation has been achieved by radioisotope phosphate labeling of proteins, sometimes with in vitro assays using recombinant proteins or with site-specific phospho-antibodies. However, the target phospho-sites and kinases responsible are often unknown, and the use of radioactive isotopes is not always desirable. Therefore, as a first step in determining the functional significance of potential phosphorylation, it is useful to show that a protein can be phosphorylated in vivo in Xenopus eggs and/or embryos. This protocol describes a nonradioactive method to visualize protein phosphorylation by exposing the protein to the egg/embryo kinase environment and then observing a difference in protein migration (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] and western blot analysis) with and without treatment with the lambda phosphatase enzyme. Subsequent investigation of the ability of site-specific phospho-mutant proteins to recapitulate the effect of phosphatase treatment can be used to explore the identity of the phosphorylated sites. Moreover, the detection of multiple bands of the protein of interest even after phosphatase treatment points to the presence of other types of posttranslational modifications.