Affiliation:
1. Bionanoscience Department Delft University of Technology Delft The Netherlands
2. These authors contributed equally to this work
Abstract
AbstractIn different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay—the GTPase Glo assay by Promega—for investigation of GTPase‐effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration‐dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Conducting GTPase Glo assaysSupport Protocol 1: Analyzing GTPase assays to correlate luminescence with remaining GTPSupport Protocol 2: Fitting GTPase assay data to obtain GTPase cycling rates