Enzyme Activity Determination
1. Prolyl Hydroxylase Domain Protein 2 (PHD2) Activity Assay


Prolyl Hydroxylase Domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is the central enzyme that controls Hypoxia Inducible Factor (HIF)-α protein levels. PHD2 is a key oxygen-sensing protein in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of the hypoxic response, Hypoxia Inducible Factor-α (HIF-α), thereby targeting HIF-α for degradation.

Assay Principle

Biotinylated peptides derived from the HIF-1α oxygen-dependent degradation domain Biotin-Ahx-DLDLEALAPYIPADDDFQL or a hydroxylated control peptide Biotin-Ahx-DLDLEALAP[OH]YIPADDDFQL are immobilized on streptavidin-coated microplate. Tissue samples are homogenized in our tissue extraction buffer. Supernatants of the tissue homogenates are mixed with proprietary reaction buffer and incubated in the microplate. Peptide hydroxylation is recognized by a rabbit antibody specific for HIF-1α [Hydroxy Pro564] peptide, and detected with a donkey anti-rabbit HRP-conjugated secondary antibody. Upon the addition of the TMB substrate, the intensity of color is proportional to the activity of PHD2 in the samples. Positive and negative controls allow for assay quality assurance.

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