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| | In Vitro Rpn11 Activity Assay |
| Description: | Fluorescence polarization assays were performed in low-volume 384-well solid black plates (Molecular Devices) in quadruplicate. The assays were performed in buffer containing 50 mM Tris-HCl pH7.5, 1 mM MgCl2, 50 μM ATP, 1 mM DTT and 0.01% NP-40. The components for the assay were added in the following sequence: (1) 5 μl compound (in 3% DMSO) at different concentrations, (2) 5 μl of diluted human 26S proteasome (Enzo Life Sciences, NY. 26S proteasome was pre-incubated with epoxomicin at room temperature for 1 h, then diluted ten-fold in 1× Assay Buffer), and (3) 5 μl of substrate (3 nM Ub4peptideOG). 100 μM Zn(cyclen)2+ was present in the titration reaction for the experiments performed with Zn(cyclen)2+. Fluorescence polarization was measured at 30 °C using a PHERAstar plate reader (BMG Labtech) with excitation at 480 nm and emission at 520 nm. |
| Affinity data for this assay | |
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