Detection of Protein Ubiquitination

Summary

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Abstract

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases.  Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Protocol

Detection of protein ubiquitination in cultured cells

1. Transfect cultured cells with plasmids expressing the protein of interest and (epitope-tagged version of) ubiquitin.
2. Make complete cell lysis buffer (2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0) with 2mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitors.
3. Lyse cells with 100 µl cell lysis buffer per plate (6 cm dish). If a larger dish is used, adjust volume accordingly.  Swirl the dish carefully to let the lysis buffer cover the entire area of grown cells.
4. Collect the cells with a cell scraper and transfer the cell lysates into a 1.5 ml eppendorf tu....

Discussion

In this presentation, we first described steps that permit detecting ubiquitin modification on a protein of interest in cultured mammalian cells. In order to detect ubiquitination specifically on the protein of interest, not on non-covalently interacting proteins, we used a stringent condition for cell lysis, immunoprecipitation and washing ^1,3.  Ubiquitination, detected by immunoprecipitation of target protein in such a harsh condition followed by anti-ubiquitin immunoblotting, is therefore likely to be .......