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A superior loading control for the cellular thermal shift assay.
Delport, Alexandré; Hewer, Raymond.
Afiliação
  • Delport A; Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, 3201, South Africa. delporta1@ukzn.ac.za.
  • Hewer R; Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, 3201, South Africa.
Sci Rep ; 12(1): 6672, 2022 04 23.
Article em En | MEDLINE | ID: mdl-35461337
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, ß-actin, heat-shock chaperone 70 and superoxide dismutase-1) are all temperature sensitive. Additionally, both APP-CTFs (α and ß) behaved similarly upon temperature exposure while APP-ßCTF 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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Contexto em Saúde: 1_ASSA2030 Problema de saúde: 1_doencas_nao_transmissiveis Assunto principal: Precursor de Proteína beta-Amiloide / Doença de Alzheimer Limite: Animals Idioma: En Revista: Sci Rep Ano de publicação: 2022 Tipo de documento: Article País de afiliação: África do Sul

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Contexto em Saúde: 1_ASSA2030 Problema de saúde: 1_doencas_nao_transmissiveis Assunto principal: Precursor de Proteína beta-Amiloide / Doença de Alzheimer Limite: Animals Idioma: En Revista: Sci Rep Ano de publicação: 2022 Tipo de documento: Article País de afiliação: África do Sul
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