Figure 3. (a) Time-course of the hydrolysis of the AE label on unligated (•) and ligated (▪) DNA substrate and (b) the dependence of signal/background of the chemiluminescent HPA for NAD⁺-dependent S. aureus ligase.

(a) Substrate or product (10 nM) in Hepes (pH 7.4) assay buffer were incubated in mild alkaline stop/selection reagent at 40 °C in a water bath or heating block. The AE label content of samples at various times was quantified by measuring the amount of chemiluminescence (RLU) generated upon reaction with chemiluminescence-generating flash reagent. Hydrolysis of the AE label in substrate by the stop/selection reagent is exponential in nature, but is essentially fully protected in product. (b) Values for signal/background ratio at regular intervals were calculated from the chemiluminescence (RLU) of unligated (•) and ligated (▪) AE-labelled DNA substrate from (a). There is an increase in the signal/background ratio over time, and a steady value of 200:1 after 60 min of incubation with stop/selection reagent was achieved.