Abstract:
:Covalent modification of proteins with ubiquitin plays an important role in a wide array of cellular processes (Hershko and Ciechanover, 1998; Pickart, 2004). For this reason an increasing number of investigators in diverse research fields are confronted with the question whether their favorite proteins are ubiquitinated. Experiments to demonstrate covalent modification with ubiquitin in vivo can be quite challenging because of low steady-state levels of the ubiquitinated forms caused by degradation by the 26S proteasome and/or highly active deubiquitinating enzymes (Dubs) that remove the ubiquitin units (Pickart and Cohen, 2004; Wilkinson and Hochstrasser, 1998). Several different methods to determine whether a particular protein is ubiquitinated have been developed (Beers and Callis, 1993; Ellison and Hochstrasser, 1991; Hochstrasser et al., 1991; Treier et al., 1994). Some of these assays were described in detail by Laney and Hochstrasser (2002). This chapter is focused on one experimental approach using expression of hexahistidine-tagged ubiquitin. It can be applied to most situations in which one suspects ubiquitination of a particular protein and has been successfully used in various organisms (Kaiser et al., 2000; Treier et al., 1994).
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Kaiser P,Tagwerker Cdoi
10.1016/S0076-6879(05)99016-2subject
Has Abstractpub_date
2005-01-01 00:00:00pages
243-8eissn
0076-6879issn
1557-7988pii
S0076-6879(05)99016-2journal_volume
399pub_type
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