Detection of protein posttranslational modifications from whole-cell extracts in saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is an ideal model organism as numerous cellular mechanisms are conserved in mammalian cells. This includes posttranslational modifications (PTMs) such as ubiquitination, sumoylation, and phosphorylation. For ubiquitination, target proteins are readily modified via a cascade reaction, which can result in various forms of ubiquitination known to be associated with numerous cellular mechanisms. Therefore it becomes imperative for researchers to detect PTMs of their favorite proteins in order to determine how the target proteins function and are regulated. However, detection of ubiquitination in vivo, as well as some other PTMs, has proven challenging for researchers due to the presence of deconjugating enzymes in the cell. This chapter describes a step-by-step protocol on how to preserve and subsequently detect PTMs of your favorite protein from budding yeast S. cerevisiae whole-cell extracts.