| Assay Method Information | |
| | Fluorescence Direct Binding Assay |
| Description: | Determination of the affinities of compounds to protein containing one or more tryptophan is measurable by monitoring the fluorescence emission in direct mode. The measurements depending on the protein available amounts are performed either manually in a cuvette on ISS-PC1 photon counting spectrofluorometer or automatically in well plates on a fluorescence plate reader device. Fluorescence titrations are performed at 20° C. in the chosen binding assay buffer by using a defined constant protein concentration against ligand concentration variations. Small aliquots of known ligand concentration solubilized in DMSO were added and the fluorescence, excited at 280 nm, was recorded at 340 nm. The fluorescence intensity was corrected for protein dilution and for the filter effect (Birdsall et al.72). The corrected fluorescence intensity was plotted against the ligand concentration and fitted using a four-parameter sigmoidal function, from which the equilibrium dissociation constant Kd was computed using the law of mass action assuming a 1:1 protein-ligand complex (Eftink, 199773).Process1) Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content2) Titration measurements of the protein against ligand by at least 12 titration steps to obtain a good s-curve fit3) Repeat the same titration measurements with the ligand alone to enable correction4) Check the stability of the protein once by titration against DMSO alone5) Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of an UV-spectrophotometer6) Use Excel template for the correction of the measured raw data7) Use GraphPad Prism software for the quadratic binding fit and the KD evaluation. |
| Affinity data for this assay | |
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