| Assay Method Information | |
| | ErbB Enzyme Assay |
| Description: | The kinases were incubated with 250 nM TK-substrate biotin (CisBio, part of cat #62TK0PEC) at 1 mM ATP along with test compounds in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 uL. Compounds were prepared as a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After 30-minute incubation at 22 C., the reaction was quenched by adding 8 uL of quench solution containing 62.5 nM Sa-XL665 and 0.25x TK-Ab-Cryptate in HTRF detection buffer (all from CisBio, part of cat #62TK0PEC). After a 1-hour incubation at 22 C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. |
| Affinity data for this assay | |
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