Application Overview

FRET-quenched protease assays are a fast, quantitative way to measure protease activity^1-2

• 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 compounds^2-3

• 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

 

^1.Peng, X et al. A nonfluorescent, broad range quencher dye for Förster resonance energy transfer assays. Anal Biochem. 388(2):220-28 (2009)

^2.Peng, X et al. Quenched near-infrared fluorescent peptide substrate for HIV-1 protease assay. Proc SPIE. 7(20):60970F.1-12 (2006)

^3.Simeonov, A et al. Fluorescence Spectroscopic Profiling of Compound Libraries. J Med Chem. 51(8):2363-71 (2008)

^4.IRDye^®Near-Infrared FRET Based Assays LI-COR Biosciences (2007)

Workflow

Technical Resources

• The Next Step in Near Infrared Fluorescence: IRDye^® QC-1 Dark Quencher

• IRDye^® Near-Infrared FRET Based Assays

• Urlacher, T et al.
  Improved Protease Detection Using IRDye^® 800RS Labeled Casein
  Poster presentation, Society of Biomolecular Sciences Annual Meeting (2006)

• Click to see a complete list of pack inserts

PubMed

Publishing with LI-COR data?

• Johnson, CE et al.
  Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring .
  Cell Death Differ. [Epub ahead of print] (2009).

• Peng, X et al.
  A nonfluorescent, broad range quencher dye for Förster resonance energy transfer assays.
  Anal Biochem. 388(2):220-28 (2009)

• Blum, G et al.
  Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes.
  Nat Chem Biol. 3(10): 668-77 (2007)

• Peng, X et al.
  Quenched near-infrared fluorescent peptide substrate for HIV-1 protease assay.
  Proc SPIE. 7(20):60970F.1-12 (2006)

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