Assay Method Information

Assay Name:  USP30 Enzymatic Assay (Rhodamine Assay)
Description:  USP30 inhibition was tested with recombinant human USP30 using the fluorescent substrate Ubiquitin-Rhodamine 110 (UBPBio) in 1536-well format. Compounds in DMSO were dispensed using a Labcyte Echo 550 (50nl/well). 2ul of 4nM USP30 in 25mM HEPES (pH 7.5) 100 mM NaCl 0.01% Triton X-100 1mM DTT was added to each well and incubated for 2hr at room temperature. 6ul of 53nM Ubiquitin-Rhodamine 110 in 25nM HEPES (pH 7.5) 100nM Nacl 0.01% Triton X-100 4mM DTT 0.02% BSA was then added to each well and incubated for an additional 2hr at room temperature. Fluorescence measurements were obtained using an Envision 2102 (Perkin Elmer) with λex=485 nm and λex=535 nm. USP30 with DMSO and buffer alone with DMSO were used for normalization for 100% activity and 0% activity, respectively.
Affinity data for this assay
 

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