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Assay Method Information |
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| | Homogeneous Time-resolved Fluorescence (HTRF) Assay |
| Description: | Enzymes were assayed with retinoblastoma substrate in 384-well plates containing diluted test compounds. Final ATP concentration was 3x the respective enzyme Km. The phosphorylated substrate was analyzed by adding lance europium anti-rabbit IgG and anti-His-allophycocyanin, resulting in fluorescence resonance energy transfer between europium anti-rabbit and allophycocyanin, and quantified by fluorescence intensity ratio 665 nm/615 nm (excited at 340 nm). IC50 values were calculated from net readings at 665 nm, normalized for europium readings at 615 nm. |
| Affinity data for this assay | |
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