Western blot of ubiquitin thioester assays for the E1 activating enzyme provided and an example E2, UbcH5a (user supplied). Procedures as described in “Assay Protocol” section. Ubiquitin-enzyme conjugates were detected by Western blotting of E1-Ub (A/B) or E2-Ub (C/D) thioester assays in the presence (A/C) or absence (B/D) of Mg-ATP co-factor, using the Ubiquitin antibody provided, as described in “Western Blot Analysis” section. Results demonstrate the formation of ubiquitin thioester linked E1 and/or E2 conjugates in all +Mg-ATP reactions. The absence of such conjugates in negative control reactions demonstrates that their formation is Mg-ATP dependent (required for E1 activation) and hence derived from the ubiquitin cascade.