Varioskan fluorescent plate reader

Protease activity against casein was tested using the EnzChek protease assay kit (Molecular probes #E6638, green fluorescent casein substrate). The working solution was prepared by diluting the stock to 10 µg/ml in 50 mM sodium citrate, pH 4.5. The purified protease fractions were diluted with sodium citrate buffer. 100 µl of the diluted substrate was combined with the diluted protease fractions in a 96 well sample plate. The plate was then covered and kept at 37 °C for 1–3 h. Fluorescence readings were taken at 1, 2, and 3 h with a Varioskan fluorescent plate reader (Thermo Scientific) using 485 nm excitation and 530 nm emission.

Protease activity against casein was tested using the EnzChek protease assay kit (Molecular probes #E6638, green fluorescent casein substrate) or the QuantiCleave protease assay kit (Pierce #23263, succinylated casein). The working stock solution was prepared by diluting the stock to 10 μg/ml in 50 mM sodium citrate, pH 4.5 or pH 5.5. The purified protease fractions were diluted with sodium citrate buffer. 100 μl of the diluted substrate was combined with the diluted protease fractions in a 96 well sample plate. The plate was then covered and kept at 37°C for one to three hours. Fluorescence readings were taken at one, two, and three hours with a Varioskan fluorescent plate reader (Thermo Scientific) using 485 nm excitation and 530 nm emission. To measure the QuantiCleave assay reaction 50 μl of TNBSA reagent was added to every well and the plate was incubated at 37°C. The absorbance at 450 nm was measured for the whole plate. Control wells with supernatant without substrate were used as background controls. The nonspecific background signal was subtracted from specific protease activity measurement.