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FRET Peptides

Custom and Catalog FRET Substrates

 

DESIGN & SYNTHESIS OF FRET PEPTIDES

   Custom and Catalog FRET Peptides
  FIGURE. FRET caspase-1 substrate, Dabcyl-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (CASP-023). This fluorogenic caspase-1 substrate enables a continuous assay of caspase-1 helpful in the screening of inhibitory compounds (Km = 11.4 µM, kcat = 0.79 s-1)
  We have a wide range of ready-to-ship FRET peptide substrates available in our store. LEARN MORE 
  For custom peptide FRET substrates please submit a QUOTATION REQUEST  

CPC Scientific has extensive knowledge in the design and synthesis of peptide FRET substrates.1-14 We offer a wide range of FRET substrates to suite your research needs as pre-manufactured FRET peptides or as custom FRET sequences. As part of our services, we provide free consulation to help you design your FRET peptide and select the appropriate FRET pair (see Table of Common FRET Pairs). We often recommend our trade-marked highly efficient quencher, CPQ2TM, to pair with the fluorecent donor 5-carboxyfluorescein (5-FAM). This efficient pair, 5-FAM/CPQ2TM, has been cited in a variety of publications in research areas spaning from cancer therapeutics to diabetes.3-12

FRET (Fluorescence Resonance Energy Transfer) is a distancedependent dipole-dipole interaction without the emission of a photon, which results in the transfer of energy from an initially excited donor molecule to an acceptor molecule. It allows the detection of molecular interactions in the nanometer range. FRET peptides are labeled with a donor molecule and an acceptor (quencher) molecule. In most cases, the donor and acceptor pairs are two different dyes. The transferred energy from a fluorescent donor is converted into molecular vibrations if the acceptor is a non-fluorescent dye (quencher). When the FRET is terminated (by separating donor and acceptor), an increase of donor fluorescence can be detected. When both the donor and acceptor dyes are fluorescent, the transferred energy is emitted as light of longer wavelength so that the intensity ratio change of donor and acceptor fluorescence can be measured. In order for efficient FRET quenching to take place, the fluorophore and quencher molecules must be close to each other (approximately 10-100 Å) and the absorption spectrum of the quencher must overlap with the emission spectrum of the fluorophore. While designing a donor-quencher FRET system, a careful comparison of the donor’s fluorescence spectrum with the quencher’s absorption spectrum is required.

The design and synthesis work at CPC for FRET and TR-FRET peptide substrates include modification of sequences, selection of donor/quencher pairs, improvement of FRET substrate solubility and quenching efficiency.

CPC has experience with a wide range of protease peptide substrates including:

  • Aggrecanase
  • ADAMs
  • ACE-2
  • APCE
  • 2A protease
  • BACE1
  • Calpains
  • Capases
  • Carboxypeptidases
  • Caspases
  • Cathepsins
  • Chymopapain
  • Complement component C1s
  • CMV protease
  • ECE-1
  • Factor Xa
  • Furin
  • Granzyme K
  • HCV protease
  • HIV protease
  • HRV1
  • Kallikreins
  • Interferon alpha A
  • Lethal Factor Protease
  • Malaria Aspartyl Proteinase
  • MMPs
  • Pepsin
  • Plasmin
  • Plasmepsin II
  • Proteinases
  • Protein Tyrosin Phosphatase
  • Renin
  • SARS
  • TACE
  • Thrombin
  • TEV protease
  • Trypsin
  • West Nile Virus Protease

TABLE OF COMMON FRET PAIRS.

Donor (Fluorophore) Excitation Emission Acceptor (Quencher)
EDANS (5-[(2-Aminoethyl) amino] naphthalene-1-sulfonic acid) 340 nm 490 nm Dabcyl (4-(4-Dimethylaminophenylazo)benzoyl)
Lucifer Yellow 430 nm 520 nm Dabsyl (4-(4-Diethylaminophenylazo)benzenesulfonyl)
Mca (7-Methoxycoumarin-4-yl)acetyl) 325 nm 392 nm Dnp (2,4-Dinitrophenyl)
Abz (2-Aminobenzoyl) 320 nm 420 nm pNA (para-Nitroaniline)
Abz (2-Aminobenzoyl) 320 nm 420 nm 3-Nitro-Tyr (3-Nitro-tyrosine)
Abz (2-Aminobenzoyl) 320 nm 420 nm 4-Nitro-Phe (4-Nitro-phenylalanine)
FITC (Fluorescein isothiocyanate) 490 nm 520 nm Dnp (2,4-Dinitrophenyl)
5-TAMRA (Carboxytetramethylrhodamine) 547 nm 573 nm QSY7
CP488 495 nm 519 nm CPQ2TM (proprietary structure)
5-FAM (5-Carboxyfluorescein) 492 nm 518 nm CPQ2TM (proprietary structure)
Cy5 647 nm 665 nm QSY21
Dansyl (5-(Dimethylamino)naphthalene-1-sulfonyl) 342 nm 562 nm 4-Nitro-Phe (4-Nitro-phenylalanine)
Trp (Tryptophan) 280 nm 360 nm Dnp (2,4-Dinitrophenyl)
Trp (Tryptophan) 280 nm 360 nm 4-Nitro-Z (4-Nitro-benzyloxycarbonyl)

CUSTOM FRET PEPTIDE CITATIONS.

  1. LaRock, Christopher N., et al. "IL-1b is an innate immune sensor of microbial proteolysis." Science Immunology 100.200: 300. (2016
  2. Goupil, Louise S., et al. "Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria." PLoS Negl Trop Dis 10.8 (2016): e0004893. 
  3. Welsh, J. D., et al. "Platelet‐targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse." Journal of Thrombosis and Haemostasis 10.11 (2012): 2344-2353. 
  4. Doron, Lior, et al. "Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease." PloS One 9.10 (2014): e111329. 
  5. Ikeda, Zenichi, et al. "Fused heterocyclic compound." U.S. Patent Application No. 14/619,489. 
  6. Ikeda, Zenichi, et al. "Heterocyclic compound." U.S. Patent Application No. 14/619,464. 
  7. Kwong, Gabriel A., et al. "Mathematical framework for activity-based cancer biomarkers." Proceedings of the National Academy of Sciences 112.41 (2015): 12627-12632. 
  8. Miles, Linde A., et al. "Seneca Valley Virus 3C pro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy." PloS One 10.6 (2015): e0129103. 
  9. Zhu, Shu, et al. "FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow." Blood 126.12 (2015): 1494-1502. 
  10. Zhu, S., et al. "Platelet‐targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow." Journal of Thrombosis and Haemostasis (2016). 
  11. Hershey, David M., et al. "Magnetite biomineralization in Magnetospirillum magneticum is regulated by a switch-like behavior in the HtrA protease MamE." bioRxiv (2016): 047555. 
  12. Dudani, Jaideep S., et al. "Sustained‐Release Synthetic Biomarkers for Monitoring Thrombosis and Inflammation Using Point‐of‐Care Compatible Readouts." Advanced Functional Materials 26.17 (2016): 2919-2928. 
  13. Shi, Jing, et al. "Properties of Hemoglobin Decolorized with a Histidine‐Specific Protease." Journal of Food Science 80.6 (2015): E1202-E1208. 
  14. Jensen, Jesper Langholm, et al. "The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay." Journal of Dairy Science 98.5 (2015): 2853-2860.