Assay Method Information

Assay Name:  Protease Inhibition Assay
Description:  HIV-1 protease was purified and refolded from E. coli inclusion bodies. The substrate used spans the p17-p24 processing site (R-V-S-Q-N-Y-P-I-V-Q-N-K) and was derivatized with biotin and fluorescein isothiocyanate (FITC) at the amino and carboxy termini. In the presence of HIV-l protease, the substrate is rapidly cleaved into two parts. The biotinylated amino terminal fragment binds to the avidin beads while the FITC-containing carboxy1 terminal fragment is removed through the membrane. In the presence of an HIV-l protease inhibitor, the substrate does not cleave and the intact peptide binds to the beads. The bound fluorescence is obtained by processing on an IDEXX Screen Machine. Determination of Ki values with this assay requires analysis under conditions in which substrate concentration resides substantially below Km and the inhibitor concentrations greatly exceeds the Ki value and the enzyme concentration.
Affinity data for this assay

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