I would like to ask two technical questions about EDANS-CO-CH2-CH2-CO-ALERMFLSFP-Dap(DABCYL)OH (Cat.No.: HY-P5326):
1. Background fluorescence characteristics:
- Does the probe have autofluorescence when it is not cleaved or chemically treated (i.e., is the intrinsic fluorescence of the EDANS group effectively quenched by DABCYL)?
2. Detection mechanism: Is its design based on the principle of fluorescence resonance energy transfer (FRET)?
- When a specific protease (such as the target protease) cuts the peptide chain (such as the ALERMFLSFP sequence), does it release the fluorescence signal by separating EDANS (donor) and DABCYL (quencher)?
Thanks for the answer!
[Troubleshooting Questions are selected from customer consultation emails.]
Plasmin I substrate peptide EDANS-CO-CH2-CH2-CO-ALERMFLSFP-Dap(DABCYL)OH (Cat.No.: HY-P5326) is a fluorescence quenching peptide substrate based on the fluorescence resonance energy transfer (FRET) mechanism, and its characteristics are as follows:
1. Molecular design principle:
- Dap (diaminopropionic acid) is used as the donor fluorophore, carrying an excitable fluorescent group;
- EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) is used as the quenching group, which inhibits the fluorescence emission of Dap through spatial proximity.
2. Background fluorescence characteristics:
When no enzyme cleavage occurs, the donor fluorophore and the quenching group form effective FRET due to tight coupling, resulting in significant suppression of background fluorescence, which only appears as a low baseline signal at the excitation/emission wavelength (Ex/Em = 340/490 nm).
3. Enzyme activation mechanism:
When the target protease cuts the specific sequence in the peptide chain (such as ALERMFLSFP), the spatial distance between the EDANS quencher and the Dap fluorophore increases, the FRET effect is released, and the fluorescence signal is significantly enhanced.
The answer to this question comes from MCE Technical Support.
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