| Assay Method Information | |
| | In Vitro Ezyme Assay |
| Description: | 5 uL of each compound dilution were robotically pipetted to Costar serocluster plates maintaining the same plate layout. All assay mixtures consisted of the following volumes: 5 uL diluted compound, 10 uL target enzyme preparation, 1 uL substrate, 5 uL assay ATP. From each assay mixture, 10 uL of assay mixture was spotted onto Millipore Multiscreen-PH opaque plates and washed twice for 10 minutes in 1% phosphoric acid. The plates were dried at 40 C. for 30 minutes, then substrate-phosphate complexes were quantitated by scintillation counting. These Millipore plates are in a 96-well format with immobilized P81 phosphocellulose membranes in the wells. Both the phosphorylated and non-phosphorylated form of the substrate bind to the membrane while ATP (unincorporated phosphate) is removed by subsequent wash steps. |
| Affinity data for this assay | |
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