| Assay Method Information | |
| | Tyrosine Kinase FGFR1 Activity Assay |
| Description: | 1. Enzyme reaction substrate μPoly(Glu,Tyr)4:1 was diluted with PBS without potassium ion (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4) to 201 μg/mL, an enzyme plate was coated at 125 μL/well, and incubated at 37° C. for 12-16 hours. The liquid from the well was discarded. The plate was washed for three times with T-PBS (0.1% Tween-20 in potassium-free PBS, 200 μL/well), 5 minutes for each time. The elisa plate was dried in 37° C. dryer for 1-2 hours.2. 49 μL of ATP solution diluted in reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added into each well, and 1 μL of the test compound was added to each well, and then 50 μL of FGFR1 kinase domain recombinant protein diluted in reaction buffer was added to initiate the reaction, and two control wells without ATP were required for each experiment. The reaction was performed on a Shaker (100 rpm) at 37° C. for 1 hour. The liquid from the well was discarded, and the plate was washed with T-PBS for three times.3. The antibody PY99 dilution (antibody diluted 1:500 in T-PBS with BSA 5 mg/mL) was added at 100 μL/well, and shaken for 0.5 hours on a 37° C. shaker. The liquid from the well was discarded, and the plate was washed with T-PBS for three times.4. The horseradish peroxidase-labeled goat anti-mouse second antibody dilution (antibody diluted 1:2000 in T-PBS with BSA 5 mg/mL) was added at 100 μL/well, and shaken for 0.5 hours on a 37° C. shaker. The liquid from the well was discarded, and the plate was washed with T-PBS for three times.5. 2 mg/mL OPD coloration solution (diluted with 0.1 M citric acid-sodium citrate buffer containing 0.03% H2O2 (pH=5.4)) was added at 100 μL/well, and reacted for 1-10 minutes at 25° C. in darkness.6. The reaction was quenched with 50 μL/well of 2M H2SO4, and the plate was read using a tunable microplate microplate reader VERSAmax at 490 nm. |
| Affinity data for this assay | |
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