| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) assay |
| Description: | In vitro B-RAF Kinase Assay. To determine iIn vitro activities of recombinant B-RAF enzyme, a Homogeneous Time-Resolved Fluorescence (HTRF) assay was performed. Inactive (unphosphorylated) 6HIS-Mek1 was utilized as a protein substrate, the phosphorylated product was detected with Eu3+ cryptate-labeled anti-phosphotyrosine PT66 antibody (Anti-Phospho Mek1/2(Ser217/221)-Cryptate, Cisbio International). Meanwhile, an Anti-6HIS-d2 antibody (Anti-6HIS-d2, Cisbio international) was added to detection system. When the two antibodies were close enough, energy, transfer was happened between Eu and d2, then activities of enzyme was determine by measuring fluorescence intensity (320 nm excitation, 665 nm emission).IC50 determination. To evaluate iIn vitro potency of compounds against B-RAF enzyme, the IC50 values of compounds of this invention were determined. Compounds were 3-fold serially diluted with 100% DMSO from 1 mM, then 4 ml of compounds were transferred to 96 ml of reaction buffer (50 mM HEPES pH7.4, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 0.005% BAS, 2 mM DTT). After mixed, 2.5 ml of 4×compound and 5 ml of 2×B-RAF of was added to a 384-well plate (OptiPlate-384, PerkinElmer), centrifuged and incubated for 5 min. Then 2.5 ml of 4×ATP (2 mM) was added to the reaction system and initiated the reaction. The assay plate was incubated in an incubator for 60 min at 23° C., then the reaction was terminated by adding 5 ml of detection solution containing Eu3+ cryptate-labeled anti-phosphotyrosine PT66 antibody, and 5 ml of Anti-6HIS-d2 antibody. |
| Affinity data for this assay | |
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