| Assay Method Information | |
| | PHD2 Enzymatic Assay |
| Description: | Compound DMSO stock preparation: All compounds were reconstituted into 20 mM stock by DMSO.Compound storage: All compounds in DMSO were stored at RT in a desiccator for short-term storage (up to 3 months). Leftover compounds were store at −20 for longer term.Working Stock Preparation:Reference Roxadustat (FG-4592) was 3-fold serial diluted from 400 μM for 10 doses in DMSO.The compounds were 3-fold serial diluted from 400 μM for 10 doses in DMSO.Prepared 200 positive control (400 μM, FG-4592) and 200 vehicle control (100% DMSO).Centrifuged compound plates at 1000 rpm for 1 min.Compound Screening:a) Transferred 40 nl compound dilutions into each well of assay plates using Echo 655;b) Sealed the assay plate and centrifuge compound plates at 1000 rpm for 1 min.c) Prepared and add 4 μL of the 2 PHD2 enzyme working solution to individual well of the assay plate.d) Sealed the assay plate and centrifuge compound plates at 1000 rpm for 1 min. Incubate plate at RT for 30 min.e) Prepared and add 4 μl 2 PHD2 substrate working solution to each well of the assay plate.f) Prepared and added 4 μL 4 stop solution to the each well of the assay plate.g) Prepared 4 detection solution with AlphaScreen Streptavidin Donor beads, AlphaScreen Protein A Acceptor beads and Hydroxy-HIF-1α (Pro564) (D43B5) XP Rabbit mAb.h) Added 4 μL 4 detection solution to the each well of the assay plate. repeat at step d.i) Read Alphascreen signal on Envision HTS plate reader. |
| Affinity data for this assay | |
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