The cellular thermal shift assay (CETSA), as a method to determine protein–ligand interaction and cellular protein modification, has rapidly become routine laboratory practice. However, current options to determine that (1) sample was loaded in each lane of the analysed western blot and (2) the amount loaded was equal, are suboptimal. Here, we report that the αC-terminal fragment of the amyloid precursor protein (APP-αCTF), detected in several wild-type mammalian cell lines, is a highly stable, soluble protein equally present from 4 to 95 °C. We demonstrate that the level of traditional loading controls (vinculin, GAPDH, β-actin, heat-shock chaperone 70 and superoxide dismutase-1) are all temperature sensitive. Additionally, both APP-CTFs (α and β) behaved similarly upon temperature exposure while APP-βCTF levels were not influenced by the presence of a binding ligand either. This emphasises that these proteins can be used as a loading control in the unlikely event of off-target binding during ligand screening. A working example is also presented for mitogen-activated protein kinase kinase in the presence of two inhibitors, PD184352 and U0126, where APP-αCTF was used to normalise the data across experimental replicates. A reduction in data variance and standard deviations was observed after normalisation. Conclusively, APP-αCTF is a superior CETSA loading control that can be used as a standard for this technique.

Current drug development strategies that target either enzymatic or receptor proteins for which specific small molecule ligands can be designed for modulation, result in a large portion of the proteome being overlooked as undruggable. The recruitment of natural degradation cascades for targeted protein removal using heterobifunctional molecules (or degraders) provides a likely avenue to expand the druggable proteome. In this review, we discuss the use of this drug development strategy in relation to degradation cascade-recruiting mechanisms and successfully targeted disease-related proteins. Essential characteristics to be considered in degrader design are deliberated upon and future development challenges mentioned.