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/) 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)