| Assay Method Information | |
| | Microscale Thermophoresis (MT) Binding Assay |
| Description: | MST (NanoTemper Technologies GmbH) was used to determine the binding affinities between Mdm2 (residues 1-118 T47W; 500 nM) and inhibitors. T47W mutation was introduced to facilitate label-free measurements and did not interfere with small molecule binding, as evidenced by unaffected affinity toward a reference compound, nutlin-3. Experiments were performed in 50 mM phosphate buffer at pH 7.4 containing 150 mM NaCl, 5 mM DTT, and 5% DMSO. Inhibitors at increasing concentration (0.763 nM to 25 uM; highest concentration was limited by solubility) were incubated with the protein for 5 min prior to measurement at 25 °C (excitation 280 nm, emission 350 nm). An inhibitor concentration-dependent decrease in tryptophan fluorescence was observed. Inhibitor binding-specific fluorescence quenching was evidenced by a loss of the effect in samples containing the inhibitor, but denatured by heating (95 °C for 5 min) in the presence of 2% SDS and 20 mM DTT. |
| Affinity data for this assay | |
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