| Assay Method Information | |
| | Fluorescence polarization assay for PKD inhibitiors-IC50 determinations |
| Description: | The PKD HTS assay was developed and run at the University of Pittsburgh Molecular Screening Center (PMLSC) as part of the Molecular Library Screening Center Network (MLSCN)(1R03DA24898-01). Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. PKD activity was assessed using an IMAP-based fluorescent polarization (FP) assay for high throughput screening (HTS) in a 384-well, small volume format. The assay procedures include (1) Add 2 uL of a substrate peptide and ATP mixture to appropriate wells. (2) Add 2 uL of a (appropriate) concentration of test compound or controls to appropriate wells. (3) Add 2 uL of PKD enzyme to each well. (4) Incubate for 1.5 hours at room temperature. (5) Add 18 uL of Binding reagent to each well and incubate for 2 hours. (6) Collect data at A485/A525. Compounds tested in three independent singlicate 10-point dose response assays, typically starting at a max. conc. of 100uM. Substance is considered inactive when IC50 is > 100 uM. |
| Affinity data for this assay | |
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