AERIUS® IMAGER
Applications for the Aerius Imaging System
PROTEASE ASSAY
Application Overview
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 digestion4-5
Suitable for protease inhibitor screening , drug candidate IC50 measurement and high throughput screening of enzyme activity
Excellent water solubility
[LEFT] 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)
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® 800RS Casein Protease Substrate LI-COR Biosciences (2007)
5.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
LI-COR Biosciences (2009)IRDye® Near-Infrared FRET Based Assays
LI-COR Biosciences (2007)IRDye® 800RS Casein Protease Substrate
LI-COR Biosciences (2007)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
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)
