| Assay Method Information | |
| | ErbB Enzyme Assay |
| Description: | In vitro enzyme assays based on CisBio HTRF-KinEASE-TK technology were used to determine compound potencies. EGFR WT (Invitrogen, cat #PR7295B) at 0.025 nM or ErbB2 V777L (Signal Chem, cat #E27-12IG-100) were incubated with 250 nM TK-substrate-biotin (CisBio, part of cat #62TKOPEC) along with test compounds in 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-35 in a final volume of 10 μL. The buffer contained 100 nM SEB (CisBio) for ErbB2 V777L, however SEB was not included for EGFR WT. The ATP concentration was at the Km for each enzyme (25 μM for EGFR WT, 110 μM for ErbB2 V777L). Compounds were prepared as a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 1 h incubation at ambient temperature, the reaction was quenched by adding 10 μL of 62.5 nM Sa-XL665 and 0.25×TK-Ab-Cryptate in HTRF detection buffer (all from CisBio, part of cat #62TKOPEC). After another 1 h incubation at ambient temperature, the extent of substrate phosphorylation was determined using a PerkinElmer EnVision multimode plate reader via time-resolved fluorescence dual wavelength detection. The percent of control (POC) was calculated using the ratio of emission at 655 nm to the emission at 620 nm. One hundred POC was determined using DMSO only controls (no test compounds present) and zero POC was determined using pre-quenched controls reactions. A 4-parameter logistic equation was fit to the POC values as a function of the test compound concentration and the IC50 value was determined as the point where the curve crossed 50 POC. |
| Affinity data for this assay | |
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