Detection of Ubiquitination in 2DE

Ubiquitination involves the tagging of proteins with one (mono-) or more (poly-) ubiquitin molecules. Primarily the role of ubiquitination involves mainly short-lived and regulatory proteins being tagged with a poly-ubiquitin tail, thus introducing a hydrophobic patch that allows the protein to be identified and degraded by the 26S proteasome. Transfer of ubiquitin to the lysine residue of a target protein is a multi-step ATP-dependent process. The functions of ubiquitination have been extended in recent years to all areas of biology, many of them proteasome independent. As a small fraction of any protein may potentially be ubiquitinated, this may explain the wide range and large number of proteins that have been identified as being tagged with ubiquitin in the literature. This chapter outlines a general method for an indication of ubiquitination levels and identification of ubiquitinated proteins by two-dimensional electrophoresis in combination with immunoblotting.