Evaluation of a potential active site inhibition of the compounds was performed using the fluorogenic Benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-MCA) substrate (Bachem Americas, Inc, Torrance, California, USA) as previously described [26 (link)]. The enzymatic activity of CatK was monitored by measuring the rate of release of the fluorogenic group, amino-methyl coumarin at an excitation wavelength of 380 nm and an emission wavelength of 450 nm using a Molecular Devices SpectraMax Gemini spectrofluorometer. Inhibitors were added prior to measurement of enzyme activity. The assays were performed at 25°C at a fixed enzyme concentrations (5 nM) and substrate concentration (5 μM) in 100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM DTT and 2.5 mM EDTA. Z-Phe-Arg-MCA hydrolysis with trypsin was carried out in 50 mM Tris-HCl, pH 8.8 at 10 nM enzyme concentration.
Spectramax gemini spectrofluorometer
Manufactured by Molecular Devices
Sourced in Italy
The SpectraMax Gemini spectrofluorometer is a laboratory instrument designed for fluorescence-based analysis. It measures the intensity of fluorescent light emitted by samples in response to excitation from a light source. The core function of the SpectraMax Gemini is to quantify fluorescence signals, which can be used to detect and analyze various biomolecules, chemical compounds, and other materials.
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Lab products found in correlation
4 protocols using spectramax gemini spectrofluorometer
1
Fluorogenic Assay for Cathepsin K Inhibition
2
Calpain-1 Activity Assay Protocol
3
Intracellular ROS Measurement Protocol
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Cells (2 × 104 cells) were seeded on 96-well plates. The cells were pretreated with each dimer (6 –8 ) at concentrations of 20 μM for 24 h and then exposed to X (0.5 mM) and XO (5 mU·mL−1) for 3 h. The cell plates were washed twice with Hanks’ Balanced Salt Solution (HBSS) and incubated in the same buffer containing 10 μM DCFH-DA for 1 h at 37 °C in the dark. Oxidation of the dye by intra-cellular ROS generates a fluorescent 2′,7′-dichlorofluorescin (DCF) signal. Intracellular fluorescence was detected using a SPECTRA max GEMINI spectrofluorometer (Molecular Devices, Rome, Italy, excitation wavelength, 495 nm; emission wavelength, 530 nm).
4
Caspase-3/7 Activity in Sebocytes
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SZ95 sebocytes were left to attach for 72 h, washed twice with PBS and switched to SFM supplemented with Ca 2+ at concentrations of 0.05, 0.2, 0.4, 0.8, and 1.4 mM for 24 h at cell confluence. Caspase-3/7 activity, a marker of cell apoptosis, was detected using a commercial assay (Apo-ONE Homogenous Caspase-3/7 Assay; Promega, Madison, WI, USA) according to the manufacturer's instructions. The released fluorescence was read on a Molecular Devices SPECTRA Max Gemini spectrofluorometer with 499 nm excitation and 521 nm emission filters. The experiment was performed twice.
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