Ubiquitin Rhodamine 110 Fluorescence Assay

The change in fluorescence was monitored for 35 min every 5 min, with excitation at 485 nm and emission at 535 nm. The total volume of the reaction mixture was 200 µL. The buffer in which the reaction took place was 20 mM Tris with 150 mM NaCl and 1 mM DTT at pH 7.5. The enzyme (10 µL) and 0.3 µL of the fluorescent substrate (Recombinant Human Ubiquitin Rhodamine 110 Protein) dissolved in DMSO were added to the reaction mixture, so that the final substrate concentration was 0.375 µM. Gramicidin D (2.5 μM, 10 μM, 40 μM, and 60 μM), dissolved in DMSO, was used as a possible inhibitor. Buffer was used as a blank, and buffer with the substrate was used as a control.