| Assay Method Information | |
| | In vitro biochemical assay for USP30 enzyme |
| Description: | The in vitro assay for USP30 evaluates the ability of a test compound to inhibit the activity of the enzyme to cleave ubiquitin from a substrate. Ubiquitin-rhodamine 110 is a quenched, fluorescent substrate for USP30. Cleavage of the amide bond between the C-terminal glycine of ubiquitin and rhodamine results in an increase in rhodamine fluorescence at 535 nm (Exc. 485 nm). While the di-substituted rhodamine moiety in Ub-Rho110-G is essentially non-fluorescent, cleavage results in a monosubstituted rhodamine, Rho110-G, which exhibits intense fluorescence when excited at 485 nm.The activity of USP30 was validated by determining the increase in fluorescence measured as a result of the enzyme catalyzed cleavage of the fluorogenic substrate Ubiquitin-Rhodamine110-Glycine generating Ubiquitin and Rhodamine110-Glycine. Incubation of the substrate in the presence or absence of USP30 was compared to confirm the deubiquitylating activity of USP30. The USP30 enzyme assay was performed by pre-incubating 20 nM of USP30 with varying concentration of a test compound in the assay buffer [PBS (pH 7.4), 1 mM DTT, 0.01% Tween 20, 0.01% BSA, DMSO final concentration is 1%] for 15 mins at room temperature, following which 100 nM of substrate Ub rhodamine was added and incubated at room temperature for 2 hrs and the plate was read for fluorescence intensity at Ex. 485 nm/535 nm in VICTOR X5 plate reader. The percent inhibition of activity of the enzyme is calculated by comparing counts in the presence and absence of compounds.Test compounds were screened at various concentrations (10-12 tested concentrations) and dose response curves were generated was generated using GraphPad Prism software Version 7 (San Diego, Calif., USA) with non-linear regression curve fit for sigmoidal dose response (variable slope). |
| Affinity data for this assay | |
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