| Assay Method Information | |
| | PLpro Inhibitory Activity Assay |
| Description: | 1. Reaction buffer: 20 mM HEPEs, pH 7.5, 100 mM NaCl, 1 mM TCEP2. Preparation of mother liquor:(1) 20 μM Ub-AMC (Ub-AMC dry powder was directly dissolved with the reaction buffer and used after centrifugation for removing precipitates);(2) 400 nM PLpro (after molecular sieve purification, frozen at −80° C., thawed on ice before use, diluted with the reaction buffer);(3) 40 μM test compound (the test compound dry powder was dissolved with DMSO to 40 mM, diluted with 50% DMSO to 400 μM, and diluted with the reaction buffer to 40 μM);3. For the reaction system of a single point inhibition test: 10 μM Ub-AMC, 100 nM PLpro, 1 μM test compound, total volume 20 μL, reacted in a 384-well plate;5 μL PLpro mother liquor+5 μL test compound mother liquor were added to a 384-well plate and incubated at 4° C. for 30 min;10 μL Ub-AMC mother liquor was added to the 384-well plate, and after the mixture was reacted at 37° C. for 30 min, the fluorescence intensity of AMC was measured (excitation: 360 nm; emission: 460 nm);4. Control group (+Control): DMSO at corresponding dilution factors replaced the test compound; blank group (Blank): Reaction buffer replaced PLpro;5. Data processing: The Blank value was subtracted from the measured value, and normalization was performed on the basis of the DMSO value;6. IC50 determination:Test compound concentration gradient (nM): 10000, 5000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 10, 5, 2, 1, 0.5, 0.1, and 0.01The fluorescence value was measured after 15 min of reaction (the enzyme reaction rate was in a linear interval around 15 min and was in a non-linear interval at 30 min);7. Data fitting: The data was normalized and processed using Sigmaplot (fitting equation: Logistic, 3 Parameter). |
| Affinity data for this assay | |
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