Introduction
For optimization of Axl/TAM receptor inhibitors, an Axl binding assay is established (HTRF method; Kinase tracer 236). This assay is based on the binding and displacement of the Alexa Fluor 647-labelled Kinase tracer 236 to each glutathione S-transferase (GST)-tagged kinase used in the binding assay. Binding of the tracer to the kinase was detected by using europium (Eu)-labelled anti-GST antibodies. Simultaneous binding of both the fluorescent tracer and the Eu-labelled antibodies to the GST-tagged kinase generates a fluorescence resonance energy transfer (FRET) signal. Binding of inhibitor to the kinase competes for binding with the tracer, resulting in a loss of the FRET signal. For the assay, the compound is diluted in 20 mM HEPES, pH 8.0, 1 mM DTT, 10 mM MgCl2 and 0.01% Brij35. Then, the kinase of interest (5 nM final concentration), fluorescent tracer (15 nM final concentration) and LanthaScreen Eu-anti-GST antibody (2 nM final concentration) are mixed with the respective compound dilutions (from 5 nM to 10 μM) and incubated for 1 h. The FRET signal is quantified using an EnVision Multilabellreader 2104.
General Information
| Cas No. | 1361030-48-9 |
|---|---|
| Purity | 98+% |
| Molecular Weight | 560.55 |
| Formula | C30H26F2N4O5 |
| Solubility | DMSO : 100 mg/mL warmed with 50ºC water bath Ethanol : 2 mg/mL warmed with 50ºC water bath Water : Insoluble |
| Storage instruction | -20°C for 3 years / -80°C for 2 years in solvent |
PREPARING STOCK SOLUTIONS
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