| Assay Method Information | |
| | Measurement of Human H-PGDS Binding Inhibitory Activity |
| Description: | The H-PGDS binding inhibitory activity of the test compound was measured by AlphaLISA antagonistic assay. A specific protocol is as follows. In an AlphaPlate-384 Shallow well plate (Cat. 6008350, PerkinElmer) were added and mixed 240 nL/well of DMSO solution as a compound solution or a solvent control, 3 L/well (final concentration: 45 nmol/L) of biotinylated H-PGDS inhibitor (see below for the production method) diluted with 2 A(v/v) DMSO-containing Assay Buffer (50 mM HEPES-NaOH (pH 7.5), 2 mM GSH, 150 mM NaCl, 2 mM MgCl2, 0.005% Surfactant P20), and 2 μL/well (final concentration: 0.3 nmol/L) of His-tagged human H-PGDS (Cat. ATGP1557, ATgen) diluted with Assay Buffer. As a blank, 2 μL/well of Assay Buffer was added instead of His-tagged human H-PGDS. This plate was allowed to stand at room temperature for 60 min, and Nickel Chelate AlphaLISA Acceptor Beads (Cat. AL108C, Perkin Elmer) at 5 μL/well (final concentration: 20 μg/mL) were added and mixed. After allowing to stand at room temperature for 30 min under shading, AlphaScreen Streptavidin donor beads (Cat. 6760002S, Perkin Elmer) were added at 5 μL/well (final concentration: 30 μg/mL) and mixed. After allowing to stand at room temperature for 60 min under shading, AlphaLISA signals (excitation wavelength: 680 nm, measurement wavelength: 615 nm) were measured using Enspire (Perkin Elmer). |
| Affinity data for this assay | |
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