Efficient approaches for characterizing ubiquitinated proteins

Post-translational modification of proteins offers a rapid route to change the activity of crucial factors within the cell. one of the more drastic post-translational modifications, in terms of effect on biochemical properties, is the covalent attachment of the small protein ubiquitin, to a target factor. The labile nature of some post-translational modifications puts obstacles in the path of attempting to detect modified species of most proteins. indeed, ubiquitination can be rapidly reversed by the action of a large family of DUBS (deubiquitinating enzymes), most of which are cysteine proteases. This, taken together with the rapid proteasomal degradation of some species of ubiquitinated proteins, results in difficulties in detecting modified targets. In this review, practical approaches developed for the detection, purification and characterization of ubiquitinated proteins are reviewed. After a brief appraisal of the use of histidine-tagged ubiquitin, focus is placed on development of UBD (ubiquitin-binding domain)-ubiquitin affinity purification.