| Assay Method Information | |
| | Enzyme-linked immunosorbent assay (ELISA) Assay |
| Description: | In House Procedure for BTK, BMX, EGFR and ITK. Kinase inhibitory activities of compounds were evaluated using the Enzyme-linked immunosorbent assay (ELISA). The kinase enzyme of BTK, BMX, EGFR and ITK were purchased from Carna Bioscience (Kobe, Japan). A total of 10 ng/mL antiphosphotyrosine (PY713) antibody (abcam, Cambridge Science Park, UK) was precoated in 96-well ELISA plates. The kinase enzymes in each reaction well were set to BTK (101.25 ng/mL), BMX (90 ng/mL), EGFR (90 ng/mL) or ITK (120 ng/mL) and incubated with indicated drugs in 1× reaction buffer (50 mmol/L HEPES pH 7.4, 20 mmol/L MgCl2, 0.1 mmol/L MnCl2, 1 mmol/L DTT) containing 20 μmol/L (the final concentration of substrate in ITK reaction was 30 μmol/L) substrate (NH2-ETVYSEVRK-biotin) at 25° C. for 1 h. Then, a total of 3 μmol/L ATP was added and the reaction was continued for 2 h. The products of reaction were transferred into 96-well ELISA plates containing antibody and incubated at 25° C. for 30 min. After incubation, the wells were washed with PBS and then incubated with horseradish peroxidase (HRP)-conjugated streptavidin. The wells were visualized using 3,3′,5,5′-tetramethylbenzidine (TMB), and chromogenic reaction was ended with 2 mol/L H2SO4, the absorbance was read with a multimode plate reader (PerkinElmer, USA) at 450 nm. IC50 values and curve fits were obtained using Prism (GraphPad Software). |
| Affinity data for this assay | |
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