Measuring Ubiquitin Conjugation in Cells

Protein ubiquitination is crucial to many diverse and critical functions of cells. Although it has been long known that conjugation of ubiquitin to proteins results in their destruction by the proteasome, recently it has become apparent that reversible protein ubiquitination, particularly monoubiquitination, performs regulatory functions in cells, analogous to protein phosphorylation. The most powerful and sensitive technique for measuring specific protein ubiquitination is antiubiquitin immunoblotting of the immunoprecipitated protein after gel electrophoresis. Efficient antibodies recognizing ubiquitinated proteins are now available, making ubiquitin immunoblotting a practical tool for research into the many and varied aspects of this extremely interesting posttranslational protein modification. Here, we describe in detail the steps to follow in order to determine whether a particular protein might become ubiquitinated, or deubiquitinated, and we offer warnings about pitfalls to avoid in antiubiquitin immunoblotting.