METHODS ENZYMOL

Mogalisetti P, Walt DR
Methods Enzymol
DOI: 10.1016/bs.mie.2016.08.017

Abstract:

Over the last decade, femtoliter arrays have been used as a simple and robust way to encapsulate and monitor the kinetics of single enzyme molecules. Encapsulating individual enzyme molecules within a femtoliter-sized reaction chamber does not require immobilization of the enzyme molecules or fluorescent tagging of the enzyme molecules, which offers the unique advantage of observing unmodified single enzyme molecules free in solution. Several fascinating details about enzyme kinetics have been revealed using these femtoliter arrays, which were unattainable from traditional ensemble experiments. Here, we discuss various considerations to take into account when developing single-molecule enzyme assays in femtoliter arrays and the advantages and disadvantages of various protocols.