| Assay Method Information | |
| | Ubiquitin-Rhodamine 110 Assay |
| Description: | Each assay was performed in a final volume of 8 μl in assay buffer containing 10 mM HEPES pH 7.3 ((1 M, pH 7.3 solution, (VWR J848), 100 mM NaCl (5 M, Corning 46-032-CV), 0.01% Triton X-100 (Sigma #T8787), 3.5 mM DTT (1 M, Sigma #43819) and 0.00375% BSA (10%, Calbiochem, #126609)). The assay buffer pH was adjusted to 7.5 using 1 N NaOH (JT Baker, #5000-03). Stock compounds were stored at −80° C. as a 25 mM in DMSO solution along with their respective 20 point 2 fold serial dilutions. For the dose responses, stock compound plates were allowed to come to room temperature the day of the assay. 10 nl of the serial dilution series were pre stamped into assay plates (Black, high base, medium binding, Greiner #782076) for a final top screening concentration of 125 μM (DMSO final concentration=0.5%). Enzyme (His6 USP28, BostonBiochem, #E-570) concentration and incubation times were optimized for the maximal signal to background while maintaining the initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was 150 pM. Final substrate (Ub-Rhol10, Ubiquitin-Rhodamine 110, UBPBio, #M3020) concentration was 41 nM. 2 μl of 4× enzyme was added to assay plates (pre stamped with compound) and incubated for 2 h at room temperature. 6 μl of 4× substrate was subsequently added to the assay plates and incubated for 2 h at room temperature. Fluorescence was then read on the Envision (Excitation 485 nm and Emission at 535 nm, Perkin Elmer). |
| Affinity data for this assay | |
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