96 multichannel pipetting head

Proteasome activity assays were performed using 5 nM of Sm20S, c20S or i20S and 25 μM of z-LLE-AMC or Suc-LLVY-AMC in assay buffer consisting of 20 mM Tris-HCl, pH 7.5, 0.02% SDS. For single concentration inhibition assays, Sm20S was preincubated with 10 nM inhibitor for 30 min at 4°C in assay buffer before the addition of z-LLE-AMC or Suc-LLVY-AMC. Assays with z-LRR-AMC were performed with 25 nM of enzyme in 20 mM Tris-HCl, pH 7.5. SDS was omitted from the z-LRR-AMC assays as it is was found to interfere with this substrate. For single concentration inhibition assays, Sm20S was preincubated with 50 nM inhibitor for 30 min at 4°C in assay buffer before the addition of z-LRR-AMC. The rate of AMC release was determined from 60 to 120 min and normalized to a DMSO control. For dose-response inhibition assays, an eight-point serial dilution of inhibitors in DMSO was acoustically transferred to 384 well assay plates using an ATS Gen 4 Plus (Biosero, San Diego, CA). After pre-incubation of enzyme with inhibitor for 30 min at 4°C, substrate was added to the assay plate using a Biomek FX^P workstation equipped with a 96 multichannel pipetting head (Beckman Coulter, Brea, CA). IC₅₀ values were calculated in GraphPad Prism 6 by normalizing activity to DMSO controls and interpolating the data using the log(inhibitor) – variable slope curve fitting algorithm.