| Assay Method Information | |
| | In Vitro JOSD1 Activity Assay & Ubiquitin-7-amido-4-methylcoumarin (AMC) Assay |
| Description: | In vitro JOSD1 activity assay was carried out with Ubiquitin-AMC assay. The Ubiquitin-AMC assay was carried out as previously described (Wernig, et al., 2008 Blood, 111(7):3751-9). Human JOSD1 protein was purified according to standard protocols. Recombinant JOSD1 was tested for activity in a Ubiquitin-AMC assay in the presence or absence of inhibitors. For this assay, 10 nM JOSD1 was pre-incubated with different concentrations of inhibitors or DMSO as a control in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.6, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 11 μM ovalbumin, 5 mM dithiothreitol (DTT). The reaction was incubated for 6 hours at room temperature prior to the addition of 2 μM Ubiquitin-AMC (Boston Biochem) substrate. The initial rate of the reaction was measured by collecting fluorescence data at one-minute intervals over a 30-minute period using a Clariostar® fluorescence plate reader at excitation and emission wavelengths of 345 and 445 nm, respectively. |
| Affinity data for this assay | |
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