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, often affecting physiological function. Traditionally, detection of protein phosphorylation has been achieved by radioisotope phosphate labelling of proteins, sometimes with in vitro assays using recombinant proteins, or by site-specific phospho-antibodies. However, target phospho-sites and kinases responsible are often unknown, and use of radioactive isotopes is not always desirable. Therefore, as a first step to determining 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 non-radioactive method to visualise protein phosphorylation by exposing the protein to the egg/embryo kinase environment and observing a difference in protein migration on SDS-PAGE and western blot with and without treatment with lamda 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, detection of multiple bands of the protein of interest even after phosphatase treatment points to the presence of other types of post-translational modifications.
Medical Research Council (MR/K018329/1)
Medical Research Council (MR/L021129/1)