Histochemical Technique 1: A General Method for Quantitative Enzyme Assays of Single Cell `Extracts' with a Time Resolution of Seconds and a Reading Precision of Femtomoles

摘要

Biochemists who study single cells have been constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods we describe will remove this limitation. In brief, nanogram tissue samples are dissected from frozen-dried tissue. The samples are `extracted' in microdroplets of assay cocktail. The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. In the development of this method, we evaluated several parameters required for optimization; the most important of these evaluations, including numerous empirically derived relationships, are reported here and in supplemental material provided with reprints.