Authors

Discussion

The Cellular Thermal Shift Assay (CETSA®) is a label-free method for measuring target engagement of compounds in intact cells and tissues as well as in their corresponding lysates/homogenates. Since its introduction, CETSA® has proven to be a very valuable tool for confirming target engagement (Martinez Molina et al., 2013). When the target of interest is known and the aim is to screen a smaller number of compounds, the western blot based CETSA® Navigate assay is often applied. For soluble cytoplasmic proteins these assays are most often straightforward to develop, but for less soluble proteins, e.g. membrane proteins, low abundance proteins, nuclear proteins, DNA-binding proteins or multi-compartment proteins, CETSA® Navigate assays can be more difficult to develop. Here we summarize some of the lessons learned with useful tips and tricks on how to tackle difficult targets in CETSA® Navigate.