Figure 2. (a) Demonstration of the linear dependence of chemiluminescence (RLU) on AE-labelled DNA substrate concentration in the absence of S. aureus DNA ligase (•) and presence of 50 pM S. aureus DNA ligase (^) and (b) kinetics of chemiluminescence (RLU) generation.
(a) A linear relationship was shown to exist for the concentration of AE-labelled DNA substrate up to 10 nM and approx. 2×105 RLU in the presence (50 pM) and absence of S. aureus DNA ligase. Reactions were carried out in volumes of 20 μl in Hepes (pH 7.4) assay buffer. Chemiluminescence generated upon addition of 20 μl of a solution of flash reagent was detected over a 2 s period. The calibration curve constructed using the closed circles in (a) was used in converting RLU into AE label concentration and hence that of substrate/product. (b) Reaction of AE-labelled DNA substrate in Hepes (pH 7.4) assay buffer with chemiluminescence-generating flash reagent results in the release of light at 430 nm. The concentration of AE-labelled substrate investigated was in the 0.1–10 nM range. In all cases, the lifetime of the emitted light was under 2 s.