Recombinant human PHD3 (EGLN3) protein was expressed in E. coli as the full-length protein (accession number NP_071356.1) with an N-terminal 6× His tag. The molecular weight of the protein is 31.1 kDa. Assay Conditions: 3 uM HIF1A (HIF-1a) peptide (DLDLEALAPYIPADDDFQL) was incubated with 300 nM PHD3 protein in 30 ul reaction system containing 20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 100 uM 2-oxoglutarate, 100 uM ascorbate and 50 uM (NH4)2Fe(SO4)2·6H2O for 2 hours at 30°C. MALDI-TOF was used for detection.
