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Assay Method Information

Assay Name:  Effect of Compounds on Activities of HIF Prolyl Hydroxylase-2
Description:  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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Last update November 1, 2007
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