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BROMOscan® Assay Process

Assay Process

  1. 1.  Assemble Assay Components

  •       E. coli expressed bromodomain labeled with DNA tag for qPCR readout

  •       Known binding ligand immobilized on a solid support

  •       Test compound or solvent control

2.  Equilibrate
3.  Wash solid support to remove unbound bromodomain
4.  Quantify bromodomain captured on solid support (qPCR)
5.  Compare captured bromodomain levels in test compound & solvent control samples

Assay Principle

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