| Assay Method Information | |
| | Factor B Binding Assay by TR-FRET |
| Description: | Table 1: Factor B binding affinity of each compound tested was determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology. 10 nM recombinant his-tagged Factor B catalytic domain, varying concentrations of inhibitors, 4 nM LANCE Eu-W1024 Anti-6×His Antibody and 100 nM TR-FRET_tool2 tracer was incubated in 1× Kinase Buffer A for 1 h. Measurement was performed in a reaction volume of 15 μL by adding 5 μL of the test compound, 5 μL of Factor B/antibody mixture and 5 μL of tracer into white opaque 384-well assay plates. The TR-FRET signal was read on a plate reader with an excitation wavelength of 340 nm and detection wavelengths of 615 and 665 nm. Binding affinity was determined for each compound by measuring TR-FRET signal at various concentrations of compound and plotting the relative fluorescence Emission Ratio (665 nm/615 nm) against the inhibitor concentration to estimate the IC50 from [Compound] vs Emission Ratio using the four parameters dose-response inhibition curve with a variable slope model in GraphPad Prism. |
| Affinity data for this assay | |
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