Western blot of Ubiquitin. Anti-Ubiquitin antibody, generated by immunization with Ubiquitin coupled to Rabbit IgG, was tested by western blot against total cell extract from yeast. Dilution of the antibody between 1:200 and 1:1,000 showed strong reactivity with Ubiquitinated proteins. In this blot the antibody was used at a 1:500 dilution incubated overnight at 4° C in 5% non-fat dry milk in TTBS. Detection occurred using a 1:2000 dilution of HRP-labeled Donkey anti-Rabbit IgG for 1 hour at room temperature. A chemi-luminescence system was used for signal detection (Roche). Other detection systems will yield similar results.