Streamlined Data Processing for Determination of Kd with "Accurate Constant via Transient Incomplete Separation" (ACTIS)

The determination of accurate equilibrium dissociation constants Kd of protein–small molecule complexes is important but challenging as all established methods have inherent sources of inaccuracy. ACTIS is a new method for Kd determination using transient incomplete separation of the complex from the unbound small molecule in a pressure-driven flow inside a capillary. ACTIS is accurate and its accuracy is invariant to variations in geometries of both the fluidic system and the flow. Furthermore, ACTIS is implemented using a simple fluidic system supporting its accuracy and providing a simple-to-follow/copy template for instrumentation. Despite the simple and robust instrumentation/acquisition, the current data processing workflow is cumbersome, time-consuming, and prone to hard-to-trace human errors therefore hindering ACTIS’ ability to become a practical reference method for Kd determination. This technical note describes a streamlined workflow for processing ACTIS data; the workflow is implemented as a set of open-source software tools called prACTISed (https://github.com/prACTISedProgram/prACTISed). These tools allow all steps of data processing to be performed in fast and straightforward fashion. These practical software tools complement the simple instrumentation serving both developers and users of ACTIS.