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How it works
PhosphoSens continuous assays utilize a simple add-and-read protocol, providing the capability for real-time, kinetic measurements, with a straight-forward, fluorescence intensity (FI) measurement.
These assays provide high accuracy and precision by allowing measurement of the initial reaction rate (measured as the slope of the initial linear region), typically during the first 30-60 minutes and include many data points, with readings performed every 30 seconds to two minutes.
This continuous assay format also allows for correction of compound autofluorescence, because this background signal does not change over time and can be corrected for by subtracting background signals (includes compound and all components of the reaction besides the enzyme) from the total fluorescence (all components including enzyme) to determine the net signal at each time point.
The continuous format is compatible with essentially any commercially available fluorescence microplate reader capable of kinetic readings, and associated laboratory automation equipment. No stop solution or quench/develop step is needed, minimizing time, while maximizing throughput.
