| Assay Method Information | |
| | LanthaScreen assay |
| Description: | TEC enzyme (LifeTech # PV3269) and Eu-anti-HIS antibody (Invitrogen # PV5596) are mixed and diluted in kinase buffer (50 mM Hepes pH 7.5+10 mM MgCl2+1 mM EGTA+0.01% Brij-35) to 3 and 6 nM, respectively. Final concentration in the assay for enzyme and antibody are 1 and 2 nM, respectively.Tracer (Kinase Tracer 178, Invitrogen # PV5593) is diluted in Kinase buffer to 3 nM. Final concentration in the assay is 1 nM.Serial dilutions log 10 from 1 mM to 3.16 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 33-fold in Kinase buffer (50 mM Hepes pH 7.5+10 mM MgCl2+1 mM EGTA+0.01% Brij-35).The assay is performed as follows: 5 μL/well of TEC enzyme and EU-anti-His antibody dilution is mixed with 5 μL/well tracer dilution and 5 μL/well of compound dilution in Kinase buffer. Final compound concentration in the assay ranged from 10 μM to 0.316 nM, with 1% DMSO final concentration in assay. Following a 2h incubation at room the TR-FRET signal at 615 nm and 665 nm is read. The ratio 665/615 was used to calculate values expressed as percentage of the difference in readout (S/N) of the controls with and without Tracer. IC50 values were determined by curve fitting of the experimental results in Dotmatics. |
| Affinity data for this assay | |
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