A real-time temperature series MICRO-TAG cell target engagement assay using Applied Biosystems QuantStudio system

Direct measurement of drug-target engagement in cells is a critical component of early drug discovery. Many existing cellular target engagement approaches require prior determination of protein melting temperatures, prolonged incubation steps, or detection formats with limited compatibility with real-time instrumentation. Here, we describe a real-time temperature-series MICRO-TAG assay that integrates fluorescence-based cellular target engagement with programmable thermal cycling on the Applied Biosystems QuantStudio Real-Time PCR System. The assay employs an RNase-based FRET substrate to generate real-time fluorescence signals that reflect ligand-induced thermodynamic stabilization of protein targets in intact cells. By implementing a ramp-hold-detect temperature program, the workflow eliminates the need for melting-temperature scouting and enables streamlined assay setup with quantitative signal acquisition across temperature series. Using MAPK1 as a representative target, we demonstrate reproducible real-time signal separation between ligand-treated and control samples and show that both single-concentration binding assessment and EC50 determination can be achieved within a single experimental run. This Application Note outlines the assay rationale, materials, protocol configuration, instrument programming, and interpretation of representative data.