Dabcyl-KTSAVLQSGFRKM-Glu(Edans) FRET Peptide Substrate – Catalog #B2026695

Dabcyl-KTSAVLQSGFRKM-Glu(Edans) is a dual-labeled fluorescent peptide designed for continuous FRET-based enzyme assays. It carries a Dabcyl quencher group at the N-terminus and the EDANS fluorophore conjugated to a C-terminal glutamic acid residue (Glu(Edans)). The peptide sequence KTSAVLQSGFRKM lies between these fluorophores. Supplied as 1 mg of lyophilized powder, this substrate is widely used to measure protease activity in real time, as peptide cleavage separates the fluorophore and quencher, resulting in a large increase in EDANS fluorescence.

Scientific Overview

FRET (fluorescence resonance energy transfer) peptides leverage the spectral overlap between a donor fluorophore's emission and an acceptor quencher's absorption. In this substrate, EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) serves as the donor fluorophore with excitation around 330 nm and emission around 480 nm. Dabcyl (4-((4-(dimethylamino)phenyl)azo)benzoic acid) is a nonfluorescent quencher whose absorption overlaps the EDANS emission spectrum. When the peptide is intact, FRET efficiently quenches EDANS fluorescence. Protease cleavage within the sequence KTSAVLQSGFRKM separates Dabcyl and EDANS, eliminating FRET and rapidly restoring fluorescence. This change can be monitored continuously in real time, making FRET peptides ideal for kinetic measurements of protease activity, inhibitor potency, and substrate specificity.

Key applications include:

• Continuous fluorescence assays for protease activity and kinetics
• High-throughput substrate specificity screening
• Drug discovery: protease inhibitor potency testing
• Real-time enzyme kinetic measurements compatible with plate readers and spectrofluorimeters
• Research into protease mechanism and cleavage preferences

Usage & Handling Guidance

Resuspend the lyophilized powder in assay buffer (phosphate, Tris, or HEPES buffer at pH 7–8 is typical) immediately before use. FRET peptide substrates are most sensitive in assays with minimal background fluorescence; perform measurements in black, low-background wells or cuvettes. Quench and fluorescence are sensitive to pH; buffer pH should match the protease's optimal pH for activity.

• Reconstitution: Dissolve in buffer to 0.1–10 μM (concentration depends on the protease and assay sensitivity desired).
• Fluorescence measurement: Excite EDANS at ~330 nm; monitor emission at ~480 nm (exact wavelengths depend on the instrument).
• Kinetics: Add enzyme to the substrate solution and follow the increase in fluorescence over time. Reaction rates are typically linear for the first 10–30% product formation.
• Storage of stock: The lyophilized substrate is stable at −20°C. Once reconstituted, working solutions should be used promptly or stored at 2–8°C for short term.

What You Get

• 1 mg of dual-labeled FRET peptide substrate, lyophilized
• Dabcyl quencher at the N-terminus
• EDANS fluorophore at the C-terminus (Glu(Edans))
• Optimized for continuous protease assays and kinetic studies
• For research use only (RUO)

Why Researchers Choose It

• Robust FRET pair with large dynamic range (on/off ratio) upon cleavage
• Enables real-time, kinetic measurements without stopping the reaction
• Simple readout: fluorescence increase is directly proportional to product formation
• Compatible with standard plate reader and spectrofluorimeter optics
• Proven design used in hundreds of published protease assays

Frequently Asked Questions (FAQ)

• What happens when the protease cleaves the substrate?
  Cleavage separates the Dabcyl quencher and EDANS fluorophore, eliminating FRET. EDANS fluorescence increases dramatically, making the reaction easy to follow continuously.
• How should I set up the assay?
  Dissolve the substrate in your assay buffer, add protease, and monitor EDANS fluorescence (Ex ~330 nm, Em ~480 nm) over time. Include negative controls (substrate alone) and positive controls (pre-cleaved substrate).
• How is this different from stopped-end assays?
  FRET substrates allow real-time, kinetic measurements. No stopping reagent or additional steps are needed; you simply follow fluorescence over time.
• What is the stock substrate concentration?
  Dissolve the 1 mg lyophilized peptide in a known volume of buffer. A 1 mM stock solution in 1 mL is common; exact concentration will depend on the peptide's molecular weight and your desired working concentration (typically 0.1–10 μM).
• Can I use this substrate for high-throughput screening?
  Yes. The assay is simple, rapid, and compatible with 96- and 384-well plate readers.

References

• Ekici OD, Zhu J, Wah Chung IY, Paetzel M, Dalbey RE, Pei D. Profiling the substrate specificity of viral protease VP4 by a FRET-based peptide library approach. Biochemistry. 2009;48(24):5753-9.
• Gratz A, Götz C, Jose J. A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease. J Enzyme Inhib Med Chem. 2010;25(2):234-9.