Single-molecule enzymatic studies have introduced ways to identify and track the distributions of individual enzyme's reaction rates that are averaged in ensemble kinetics. We describe a simple method that combines the use of a microfabricated array of wells, a protease assay, and fluorescence microscopy to study our model enzyme alpha-chymotrypsin at the single-molecule level. The microfabrication process for the silicon wafer mold is outlined in detail, and the enzymatic assay is compared between ensemble and single-molecule experiments. In our method, PDMS wells are designed 2mum in diameter and 1.35mum in height. We isolate alpha-chymotrypsin into single molecules inside the cylindrical wells with an enzyme-to-substrate ratio of 1: 6,666 molecules. This method allows for the simultaneous tracking of hundreds of individual enzyme molecules over time. In our analysis of single-enzyme kinetics, we incorporate orange fluorescent microspheres (540/560nm) as the reference standard and correct for photobleaching of the dye in the protease assay. Our results show that single alpha-chymotrypsin molecules exhibit heterogeneous product formation rates that are stable for long durations.
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