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. 2021 Oct 19;2(4):100896. doi: 10.1016/j.xpro.2021.100896

Figure 1.

Figure 1

General workflow for primary and dose-response screens

Prior to beginning the screening process, DUB and ubiquitin-rhodamine (Ub-Rho) solutions are prepared in assay buffer and compound source plates are prepared from DMSO stocks. Step 1: Compounds are added from source plates to 1536 well assay plates in single concentrations (for primary screen) or in either 4 or 8-point dose-response (for dose-response screens). Columns 45–46 contain DMSO only as a neutral control. Step 2: A 2x solution of DUB in assay buffer is then added to all wells on the plate except columns 47–48 (which receive only assay buffer). Step 3: A 2x solution of Ub-Rho is then added to all wells on the plate. Step 4: Plates are then incubated to allow the DUB enzymatic reaction to occur. Step 5: Fluorescence measurements for each plate are then recorded using a fluorescence plate reader followed by data analysis.