A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Prepare the appropriate assay buffers for the proteases as described in the text protocol, and chill the buffers on ice. Place a solid black polystyrene non-treated flat-bottom 96-well plate on ice with a thin metal plate underneath to support cooling and stability.

It is essential to use a Black plate as the assay plate to prevent fluorescent leakage from adjacent wells.

Per peptide, prepare three technical replicates per assay in a total volume of 100 microliters per sample. Pipette the appropriate amount of assay buffer into each well of the assay plate. Add 0.5 microliters of protease to each well. To six wells, add 0.5 microliters of buffer instead of the respective protease. Three of these will be blank controls, and the other three will be peptide controls.

Add 5 microliters of the peptide to a final concentration of 50 micromolar to each well, except the blank controls. To each of the three blank control wells, add 5 microliters of buffer instead of peptide. Insert the plate into the fluorescence plate reader and click Start.