Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins.

ABSTRACT: Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [¹⁴C]O₂ release from labeled [¹⁴C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with conce

SUBMITTER: Wong SJ 

PROVIDER: S-EPMC7961094 | biostudies-literature | 2021 Jan-Jun

REPOSITORIES: biostudies-literature