| Assay Method Information | |
| | HIV Protease Enzyme Inhibition (PI) Activity Assay |
| Description: | Inhibitor potency against HIV protease was measured using an enzymatic assay with a fluorogenic readout. To a reaction buffer containing 100 mM ammonium acetate at pH 5.3, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.25 mg/mL BSA and 1% DMSO were added 10 nM of recombinant HIV protease (concentration based on protein monomer) and test compound at one of various concentrations. After a 10-minute pre-incubation, the enzymatic reaction was initiated by the addition of the fluorogenic substrate (2-aminobenzoyl)Thr-Ile-Nle-(p-nitro)Phe-Gln-Arg (Bachem) (SEQ ID NO: 1) to a final concentration of 40 μM. The total volume of the assay solution was 100 μL. The reaction was measured over 10 minutes on a Tecan Infinite M1000 plate reader using an excitation wavelength of 320 nm and a detection wavelength of 420 nm. The slopes of the progress curves were the measure of reaction rates. Reaction rates were plotted as a function of inhibitor concentration, and the data were fit with a four-parameter logistic fit using Graphpad PRISM software to yield IC50 values. |
| Affinity data for this assay | |
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