| Assay Method Information | |
| | Biacore Assay |
| Description: | The evaluation of the binding affinity (KD) of the compounds of formula (I) to the HSP90 protein was done using Biacore T100.His-HSP90 N-term domain was immobilized by capture of his tag by the Ab previously covalently bound to the chip (CM5) surface. A short cross-linking procedure after HSP90 binding was necessary in order to obtained a more stable signal.The compounds were analyzed using the following running buffer: 20 mM Tris/HCl pH 7.6, 150 mM KCl, 5 mM MgCl2, 0.05% P20 and 1% DMSO. The flow rate was 50 ul/min with and association time of 60 seconds.The cpds were diluted in running buffer from 100x stock solution; the different conc injected were obtained from a sequential dilution in order to have the same DMSO conc. in each sample and avoid any solvent correction.A series of 5 sequential injections of increasing concentrations of compound were performed and the results were analyzed using a method reported in literature (Robert Karlsson et al). |
| Affinity data for this assay | |
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