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A superior loading control for the cellular thermal shift assay.

Alexandré Delport1, Raymond Hewer2

  • 1Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, 3201, South Africa. delporta1@ukzn.ac.za.

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Summary

Amyloid precursor protein alpha-C-terminal fragment (APP-αCTF) is a superior loading control for cellular thermal shift assays (CETSA). This stable protein normalizes western blot data, reducing variance in ligand screening experiments.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Cellular thermal shift assay (CETSA) is a standard method for studying protein-ligand interactions.
  • Current western blot loading controls for CETSA are temperature-sensitive and suboptimal.
  • Reliable loading controls are crucial for accurate CETSA data normalization.

Purpose of the Study:

  • To identify and validate a superior loading control for CETSA.
  • To evaluate the stability and suitability of amyloid precursor protein fragments as loading controls.
  • To demonstrate the effectiveness of APP-αCTF in normalizing CETSA data.

Main Methods:

  • Investigated the thermal stability of APP-αCTF and traditional loading controls (vinculin, GAPDH, β-actin, HSP70, SOD1) across a temperature range (4-95°C).
  • Assessed the impact of ligand binding on APP-CTF levels.
  • Utilized APP-αCTF as a loading control for western blot analysis in a CETSA experiment involving MAPK kinase inhibitors (PD184352, U0126).

Main Results:

  • APP-αCTF is highly stable and soluble across a wide temperature range (4-95°C).
  • Traditional loading controls exhibited temperature-dependent variations in levels.
  • APP-αCTF effectively normalized western blot data, reducing experimental variance and standard deviation in CETSA.
  • APP-βCTF levels were unaffected by ligand binding, suggesting its utility as well.

Conclusions:

  • APP-αCTF is a superior and reliable loading control for CETSA.
  • The use of APP-αCTF enhances the accuracy and reproducibility of CETSA experiments.
  • APP-αCTF can serve as a standard loading control for the CETSA technique.