Figure 2

Competitive Western blot demonstrating specificity of anti-ubiquitin antibodies. Western Blot of synthetic ubiquityl-calmodulin conjugates with an affinity purified anti-ubiquitin antibody in presence of monomeric unconjugated ubiquitin (5 mg/ml endconcentration) and ubiquitylprotein-isopeptidase. The addition of monomeric unconjugated ubiquitin led to the loss of signal from ubiquitin as well as multiubiqutin chain, attached to target protein. Reaction mixtures for uCaM-isopeptidase tests (lane 3) contained in a total volume of 230 μl 50 mM Tris/HCl, 5 mM Mg acetate, 8.5 μg uCaM-conjugates and 50 μg enriched uCaM-isopeptidase fraction. Incubation was stopped after 60 min. by adding TCA at final concentration of 5%. The pellets were neutralized with 60 μl Laemmli sample buffer, heated to 96°C for 10 min and then subjected for SDS-PAGE. First- and second order ubiquitin-calmodulin conjugates (uCaM I and uCaM II) are indicated on the left side of the panel. Lane 1: 8.5 μg ubiquityl-calmodulin conjugates. Lane 2: 8.5 μg ubiquityl-calmodulin conjugates and 5 mg/ml unconjugated ubiquitin. Lane 3: 8.5 μg ubiquityl-calmodulin conjugates incubated with 50 μg ubiquitylprotein-isopeptidases.