Automated IC50 Determination Using PULSEspencer

This application note presents a technical report on the use of the PULSEspencer series and qTOWER iris 384 for automated IC50 determination in enzyme kinetic studies. The objective is to address the challenges associated with traditional manual or semi-automated serial dilutions, which can introduce significant errors in inhibitor titrations. The report details the implementation of a continuous fluorometric protease assay utilizing trypsin as the target enzyme and benzamidine as the competitive inhibitor. It explains how the PULSEspencer R enables non-contact direct titration in miniaturized volumes, thereby minimizing reagent consumption and error propagation. The document outlines the preparation of reagents, the optimization of assay conditions, and the automated dispensing process, which includes the use of software tools for designing concentration matrices and ensuring consistent DMSO concentrations across wells. Continuous fluorescence monitoring is conducted using the qTOWER iris 384, allowing for real-time data collection and analysis of the kinetic responses, ultimately facilitating the rapid generation of precise dose-response curves.