| Assay Method Information | |
| | In vitro Hydroxylation Assays for PHD2 (AlphaScreen) |
| Description: | Inhibition assays were carried out in 384-well white ProxiPlates(PerkinElmer) in 10 μL of reaction volume. Standard reaction mixturesconsisted of the compound (in 2% DMSO final concentration), enzymemix (0.001 μM of PHD2, 10 μM of Fe(II), 100 μM of ascorbate) andpeptide mix (0.06 μM of biotinylated C-terminal oxygen dependentdegradation domain (CODD) peptide, 2 μM of 2OG) in 50 mMHEPES pH 7.5, 0.01% Tween-20 and 0.1% BSA buffer. Compoundswere preincubated with the enzyme mix for 15 min before beingincubated with peptide mix for 10 min at 22 °C. Each reaction wasquenched with 5 μL of 30 mM EDTA. |
| Affinity data for this assay | |
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