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Volume Scaling of the Transcreener® ADP2 FP Assay on the BioTek Synergy™ 2 and 4 Multi-Mode Microplate Readers Allows For Easy Transition From Assay Development to High-Throughput Screening下載
February 02, 2009
Authors: Tracy Worzella, Brad Larson, Karen Kleman-Leyer, BellBrook Labs; Xavier Amouretti, Peter Banks, BioTek Instruments
We present data showing the validation of BellBrook Labs’ Transcreener® ADP2 FP Assay on the BioTek Synergy™ 2 and Synergy™ 4 plate readers. The Transcreener ADP2 Assay is a universal, second generation far red fluorescence polarization assay that detects the ADP nucleotide by-product from any ATP-utilizing enzyme reaction. The assay is amenable to volume scaling, as shown by data generated in 96-well and 384-well microplate formats. Data generated with a standard curve in both microplate formats resulted in Z´ values ≥ 0.6. Further, the 96-well assay using the standard PMT and the 384-well assay using the red-shifted PMT meet BellBrook Labs' Instrument Validation Program criteria, which include Z´ > 0.7 with Δ mP shift ≥ 95 at 10% conversion of 10 μM ATP to ADP. Further enzyme studies demonstrate that 96- and 384-well plate formats yield excellent Z´-factor results with Protein Kinase A (PKA) at low % ATP conversion. Known PKA inhibitors show comparable potency in both 96- and 384-well plate formats, and between different Synergy plate readers. Although the standard PMT Synergy does not meet validation criteria in 384-well plate format, comparable inhibitor potency results are obtained between the standard and red-shifted PMT instruments.