A superior loading control for the cellular thermal shift assay

Sci Rep. 2022 Apr 23;12(1):6672. doi: 10.1038/s41598-022-10653-7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alzheimer Disease* / metabolism
  • Amyloid beta-Protein Precursor* / metabolism
  • Animals
  • Cell Line
  • Ligands
  • Mammals / metabolism
  • Mitogen-Activated Protein Kinase Kinases

Substances

  • Amyloid beta-Protein Precursor
  • Ligands
  • Mitogen-Activated Protein Kinase Kinases