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Protease Assay

protease figure 1

Figure 1. FRET-quenched protease assays are a fast, quantitative way to measure protease activity1-2

  • Intact substrate is fluorescence-quenched, either by self-quenching (as shown in schematic) or by a quencher dye
  • Cleavage causes dyes to separate, relieving quenching and generating signal
  • No interference from uncleaved substrate

Advantages of IRDye protease assays:

  • Simplicity — just mix and read
  • Flexibility — no stop solution required; easy to monitor reactions over time
  • Sensitivity — near-infrared detection reduces background, scattering and interference caused by other compounds2-3
  • Subnanomolar enzyme detection2
  • Large fluorescence intensity enhancement upon digestion4
  • Suitable for protease inhibitor screening, drug candidate IC50 measurement and high throughput screening of enzyme activity
  • Excellent water solubility
Inhibition of HIV-1 protease by Pepstatin A

Figure 2. Inhibition of HIV-1 protease by Pepstatin A. Proteolysis of quenched substrate (1 µM) by HIV-1 protease (20 nM) was inhibited by the addition of Pepstatin A (4.11 nM to 9 µM). After 1 hr at 37 °C, the reactions were stopped and fluorescence intensity of IRDye® 800CW measured with an Aerius Automated Infrared Imaging System. Taken from: The Next Step in Near Infrared Fluorescence: IRDye QC-1 Dark Quencher LI-COR Biosciences (2009)

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