![]() For protein–ligand binding in particular, this assay provides unique advantages over other thermal shift assays as engagement occurs intracellularly where additional pharmacological properties (such as ligand permeability and toxicity) may also be inferred. When a protein is stabilised, for example in the presence of a binding ligand 3, through protein modifications 4 or protein–protein interactions 5, 6, a discernible shift (generally an increase) in the protein Tagg is observed. By quantifying the amount of remaining soluble protein at each exposed temperature, distinct melting curves can be generated and a point where 50% of the protein remains soluble-the aggregation temperature (Tagg) (or sometimes melting temperature (Tm))-can be determined 2. The assay is based on the principle that a protein will unfold and aggregate when exposed to increasing temperature, thereby moving from a soluble to an insoluble state 2. The cellular thermal shift assay (CETSA) has become a popular method to determine a change in protein stability in a cellular environment, with over 160 experimental articles describing this technique having been published following its introduction in 2013 1. Conclusively, APP-αCTF is a superior CETSA loading control that can be used as a standard for this technique. A reduction in data variance and standard deviations was observed after normalisation. 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. This emphasises that these proteins can be used as a loading control in the unlikely event of off-target binding during ligand screening. 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. 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. 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 ☌. 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. ![]() The cellular thermal shift assay (CETSA), as a method to determine protein–ligand interaction and cellular protein modification, has rapidly become routine laboratory practice. ![]()
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