| Assay Method Information | |
| | Tec, Txk, and ITK Activities Inhibition Assay |
| Description: | The inhibitory activities of the compound to Tec, Txk, and ITK of human were performed by Eurofins Pharma Discovery Services UK Limited (UK) of the United Kingdom. In the case of Km ATP, the radioactive protein kinase method was adopted by the test. The kinase was diluted with buffer solution (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1% 6-mercaptoethanol, 1 mg/mL BSA) before the reaction. The compound was dissolved in 100% DMSO and was of 50-fold final reaction concentration.ITK reaction solution included 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.33 mg/mL myelin basic protein, 10 mM magnesium acetate, and [γ-33P]-ATP (about 500 cpm/pmol), 200 μM ATP-Mg was added, after mixing, the reaction was started, the incubation was performed for 40 minutes at room temperature, and 3% phosphoric acid solution was added to terminate the reaction. 10 μl (reaction solution) was placed on P30 filtration membrane, washed with 75 mM phosphoric acid for 5 minutes, and dried with methanol for one time, and then scintillation counting was performed.Active type Tex reaction solution contained 8 mM MOPS pH 7.0, 0.2 mM EDTA, 1 mM Na3VO4, 5 mM Na-6-phosphoglycerol, 400 μM EFPIYDFLPAKKK, 10 mM magnesium acetate, and [γ-33P]-ATP (about 500 cpm/pmol), 120 μM ATP-Mg was added, after mixing, the reaction was started, the incubation was performed for 40 minutes at room temperature, and 3% phosphoric acid solution was added to terminate the reaction. 10 μl (reaction solution) was placed on P30 filtration membrane, washed with 75 mM phosphoric acid for 5 minutes, and dried with methanol for one time, and then scintillation counting was performed.TxK reaction solution contained 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM GEEPLYWSFPAKKK, 10 mM magnesium acetate, and [γ-33P]-ATP (about 500 cpm/pmol), 200 μM ATP-Mg was added, after mixing, the reaction was started, the incubation was performed for 40 minutes at room temperature, and 3% phosphoric acid solution was added to terminate the reaction, 10 μl (reaction solution) was placed on P30 filtration membrane, washed with 75 mM phosphoric acid for 5 minutes, and dried with methanol for one time, and then scintillation counting was performed. |
| Affinity data for this assay | |
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