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KINOMEscan® Assay Process & Data Quality Metrics

Assay Process

1.  Assemble Assay Components

  •       E. coli or mammalian cell-expressed kinase labeled with DNA tag for qPCR readout
  •       Known active site binding ligand immobilized on a solid support
  •       Test compound or DMSO control

  1. 2.  Equilibrate

  2. 3.  Wash solid support to remove unbound kinase

  3. 4.  Quantify kinase captured on solid support (qPCR)

  4. 5.  Compare captured kinase levels in test compound and DMSO control samples

Assay Principle

Compounds that bind the kinase active site and directly (sterically) or indirectly (allosterically) prevent kinase binding to the immobilized ligand, will reduce the amount of kinase captured on the solid support (Panels A & B). Conversely, test molecules that do not bind the kinase have no effect on the amount of kinase captured on the solid support (Panel C). Screening “hits” are identified by measuring the amount of kinase captured in test versus control samples by using a quantitative, precise and ultra-sensitive qPCR method that detects the associated DNA label (Panel D). In a similar manner, dissociation constants (Kds) for test compound-kinase interactions are calculated by measuring the amount of kinase captured on the solid support as a function of the test compound concentration.