ADP is generated by a variety of enzymes such as kinases, ATPases or DNA helicases and therefore serves as an indicator for their enzymatic activity. The Transcreener® ADP2 FI assay by BellBrook Labs is based on Alexa594-coupled ADP which is bound to a quencher. Enzyme-generated, unlabeled ADP displaces the fluorophore-coupled ADP from its quencher and therefore increases the fluorescence intensity.
The performance of BMG LABTECH multi-mode plate readers in reading the assay was tested by measuring 384-well plates loaded with a dilution series of ADP. A final volume of 20 µl was reached after addition of the detection mix.
As expected, higher ADP concentrations resulted in increasing fluorescence intensities irrespective of reading on FLUOstar® Omega, CLARIOstar® or PHERAstar® FS. Furthermore all of the readers provided Z' factors of >0.8, verifying a robust assay. The dedicated high-throughput reader PHERAstar provided the best Z' factors and highest speed.