| Assay Method Information | |
| | Pharmacological Activity Assay |
| Description: | 1. Effect of Compounds on Activities of HIF Prolyl Hydroxylase-2 Activities of HIF prolyl hydroxylase was determined according to the method as described in Anal Biochem, 2004, 330: 74-80, which was slightly modified. A 96-well plate was pretreated with blocker casein and 1 mM biotin for 30 minutes, and then biotin-linked HIF-1α556-574 (biotinyl-DLDLEMLAPYIPMDDDFQL) was immobilized on the 96-well plate. The 96-well plate was then filled with an appropriate amount of HIF-PHD2-containing buffer (20 mM Tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl2, 20 mM 2-oxoglutarate, 10 mM FeSO4, 2 mM ascorbic acid, 4% EDTA-free protease inhibitor) and was incubated for 1 to 60 minutes at RT. The reaction mixture also contained different concentrations of HIF prolyl hydroxylase inhibitors to be tested. The reaction was stopped by rinsing the 96-well plate three times with washing buffer. In 100 μl binding buffer (50 mM tris(hydroxymethyl)-aminomethane, pH 7.5, 120 mM NaCl), hydroxylated HIF-1α556-574 was reacted with Eu-VBC protein in the binding buffer at RT for 60 minutes. The reaction solution was aspirated, and the unbound Eu-VBC protein was washed away by washing 3 times with an elution buffer. Subsequently, 10 μl of rabbit anti-Eu-VBC polyclonal antibody was added. After further 30 minutes, 10 μl of anti-rabbit polyclonal antibody immunoglobulin coupled to horseradish peroxidase was added to the binding buffer. To determine the amount of bound Eu-VBC protein, it was incubated with TMB for 15 minutes. The color reaction was terminated by addition of 100 μl of 1 M sulfuric acid. The amount of bound Eu-VBC protein was determined by measuring optical density at 450 nm, which was proportional to the amount of hydroxylated proline in the peptide substrate. |
| Affinity data for this assay | |
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