Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

Fast, robust, and inexpensive screening methods are the heart of drug discovery processes. Moreover, it is useful to have access to several established assay formats, for validation purposes. If a targeted protein is an enzyme, the logical and widely used approach is the direct measurement of the effect of the added ligands on its activity. A variety of enzymatic assay formats have been successfully applied for inhibitor screening of protein kinases. However, enzymatic assays require an active enzyme with a known substrate and often time-consuming assay optimization. Several alternative approaches have been recently developed that detect binding of ligands to proteins. This chapter overviews and provides the experimental protocol of the successful application of differential scanning fluorimetry (DSF) in our laboratory for fast and robust screening of medium-sized (<10,000) inhibitor libraries. DSF monitors the thermal stabilization of the native protein structure upon ligand binding. It allows selectivity profiling of any protein kinase without prior knowledge of either substrate or activity of the kinase under investigation. Comparative studies revealed that generated data is highly reproducible and correlates well with the results from other ligand binding methodologies, direct binding constants as well as enzymatic assays.

Original publication




Journal article


Methods Mol Biol

Publication Date





109 - 118


Enzyme Assays, Fluorometry, High-Throughput Screening Assays, Ligands, Protein Binding, Protein Kinase Inhibitors, Protein Stability, Substrate Specificity