Assay Method Information
Assay Name:  PHD1, PHD2, PHD3 Enzyme Assay
Description:  VHL/elongin B/elongin C were constructed, expressed and purified, and VHL was labeled. PHD1, PHD2 and PHD3 were constructed, expressed and purified. VBC protein was labeled with DELFIA Eu-Labeling Kit. A NeutrAvidin 96-well plate was blocked after the addition of 200 μL Blocker Casein to each well, followed by incubation for 30 min. Each well was washed 3 times by adding 200 μL of washing buffer each time. A 200 nM HIF-1a 556-574 solution was prepared with washing buffer, and 100 μL of the solution was added to each well, followed by incubation for 60 min. Each well was washed 3 times by adding 200 μL of washing buffer each time. A 1 mM Biotin solution was prepared with Blocker Casein, and 100 μL of the solution was added to each well, followed by incubation for 30 min. Each well was washed 3 times by adding 200 μL of washing buffer each time. A 20× compound solution was prepared with PHD reaction buffer, with the final concentration of DMSO being 1%. PHD solutions were prepared with PHD reaction buffer, and the concentrations of PHD1, 2, and 3 solutions were 10 ng/μL, 5 ng/μL, and 15 ng/μL, respectively. 95 μL of the PHD solution was added to each compound well, followed by the addition of 5 μL of the compound solution, 95 μL of the PHD solution was added to each full active well, followed by the addition of 5 μL of the PHD reaction buffer; and 100 μL of the PHD reaction buffer was added to each blank well. After mixing homogeneously, the wells were incubated for 60 min. Each well was washed 3 times by adding 200 μL of washing buffer each time A 1 ng/μL Eu-VBC solution was prepared with Eu-VBC binding buffer, and 100 μL of the solution was added to each well, followed by incubation for 60 min. Each well was washed 6 times by adding 200 μL DELFIA Wash Concentrate each time. 100 μL DELFIA Enhancement Solution was added to each well. A microplate reader was used to read TR-Fluorescence at Ex340 and Em615. The flurescence of the sample wells was recorded as FLUsample, the flurescence of the full active wells was recorded as FLU100%, and the flurescence of the blank well was recorded as FLU0%.
Affinity data for this assay
 
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