MV4‐11, Panc‐1, or A549 cells were harvested and collected into a tube (1 × 106 cells/tube, n = 3). The cells were lysed with lysis buffer containing 1% Triton X‐100, 20 mM Tris–HCl, 137 mM NaCl, 10% Glycerol, and 1 mM EDTA. Then, the cell lysate was collected via centrifugation (10 000×g, 4°C, 10 min). The protein concentrations in the lysate were measured using a DC Protein Assay Kit (Bio‐Rad). The activity of proteases (trypsin, chymotrypsin, thermolysin, proteinase K, proteinase XIV, and elastase) in the resultant cell lysates was determined using an Amplite Universal Fluorimetric Protease Activity Assay Kit (AAT Bioquest).
Amplite universal fluorimetric protease activity assay kit
Manufactured by AAT Bioquest
The Amplite Universal Fluorimetric Protease Activity Assay Kit is a fluorescence-based assay designed to detect and measure protease activity. The kit provides a convenient and sensitive method for quantifying the activity of various proteases in a microplate format.
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2 protocols using amplite universal fluorimetric protease activity assay kit
1
Proteases Activity Profiling in Cell Lysates
2
Fluorescent Assay for Betaxanthins and Proteinase A
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Fluorescence measurements of culture supernatants were made using a microplate reader (Fluoroskan Ascent FL - Thermo Labsystems). The following settings were used for both the betaxanthins and the proteinase A fluorescence measurements: excitation at 485 nm and emission at 520 nm. For the betaxanthins measurements, 300 μL of supernatant were transferred to a 96-well clear flat-bottom microplate (Greiner Bio-One, UV-Star®). For the proteinase A measurements, an Amplite® Universal Fluorimetric Protease Activity Assay Kit (AAT Bioquest) was used to quantify the amount of functional proteinase A in 100 μL of supernatant. Three replicates were used. Data were plotted using Prism (GraphPad).
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