Protocol for quantifying cellular drug-target engagement across a temperature series using the MICRO-TAG enzyme...

Here, we present a protocol to quantify cellular target engagement across a programmable temperature series using the MICRO-TAG split-RNase S complementation assay on a real-time quantitative PCR instrument. We describe steps for expressing target proteins as S-tag fusions in mammalian cells, applying thermal challenge and compound treatment, and measuring RNase-mediated cleavage of an RNA fluorescence resonance energy transfer (FRET) substrate. We then detail procedures for analyzing fluorescence signals across temperatures and compound concentrations, followed by four-parameter logistic fitting to derive EC₅₀ values. For complete details on the use and execution of this protocol, please refer to Babic et al.¹.