Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the heart muscle extract Figure 7a shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg heart muscle-extract and 25 μg ubiquitylprotein-isopeptidase (see material). The batches incubated with ubiquitylprotein-isopeptidases had a final concentration of 50 mM Tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. Both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.