| Assay Method Information | |
| | KD Determination by SPR |
| Description: | Affinity and Reversibility -The binding affinity and kinetics of binding were measured using Surface Plasmon Resonance based binding assay. These experiments were carried out on Bruker SPR MASS-1 and MASS-2 instruments. There was no significant difference in results obtained on both these instruments. Bap-tagged TTR protein was captured on a Streptavidin coated sensor chip to achieve about 2000 to 3000 RUs of surface density. All the samples were prepared in buffer consisting of 10 mM Sodium Phosphate, pH 7.6, 100 mM KCI, 0.005% Tween-20 and 2% DMSO. The same buffer was used as the running buffer during the experiments. Compound samples were injected at a flow rate of 30 pL/min for 90 seconds of association time followed by at least 240 seconds of dissociation period. The compounds were tested in a concentration series consisting of at least 6 samples (usually 10) made with 5-fold, 3-fold, or 2-fold dilution. The highest concentration was 10 mM or selected based on compound binding affinity observed in a previous experiment. Multiple blank injections were run before and after each compound series to allow double reference subtraction during data processing and analysis. Tafamidis or another compound with >10 replicates was tested in every experiment as a positive control to assess activity of the captured protein on the surface. A DMSO curve was run during each experiment to properly correct for excluded volume. The data were processed and analyzed using Bruker Analyzer and Scrubber to calculate binding affinities by fitting the data to 1:1 binding model. The binding parameters obtained for tafamidis binding to TTR (n=24) are listed below. Tafamidis binds to TTR in a reversible manner with calculated residence time of around 40 seconds. |
| Affinity data for this assay | |
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