| Assay Method Information | |
| | Effect of Compound I on CSF1R Kinase Activity |
| Description: | 1. ELISA Assay: The enzyme reaction substrate poly(Glu, Tyr)4:1 was diluted to 20 μg/mL with potassium-free PBS (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4). Plates were coated with the dilution, allowed to incubate at 37° C. for 12-16 h, washed with T-PBS (0.1% Tween-20 in potassium-free PBS), and dried for later use. An ATP solution (final concentration of 5 μM) diluted with a reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT), a test compound or solvent control, and a recombinant CSF1R kinase were added sequentially to individual wells to initiate the reaction. After reaction at 37° C. for 1 hour, the plates were washed with T-PBS, and the antibody PY99 dilution was added. The plates were incubated on a shaker at 37° C. for 0.5 h, and then washed with T-PBS. A horseradish peroxidase-labeled goat anti-mouse secondary antibody dilution was added, and the plates were incubated at 37° C. for 0.5 h. After the plates were washed, an OPD developing solution (diluted with 0.1 M citric acid-sodium citrate buffer (pH=5.4) containing 0.03% H2O2) was added at 2 mg/mL and the plates were allowed to react in the dark at 25° C. for 1 to 10 minutes. Finally, 2 M H2SO4 was added to stop the reaction. The plates were read with an adjustable wavelength microplate reader at a wavelength of 490 nm. The inhibition rate was calculated as follows.InhibitionRate%=(1‐CompoundODvalue‐Enzyme‐freecontrolODvalue)SolventODvalue‐Enzyme‐freecontrolODvalue×100%The IC50 values were calculated by a four-parameter regression program in the software embedded in the microplate reader. |
| Affinity data for this assay | |
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