| Assay Method Information | |
| | Fluorescent Polarization Competition Assay |
| Description: | Competition assays were performed in FP buffer (DTT was avoided for Cys-PTHRct-9) containing fixed concentrations of both the fluorescently labeled peptide andprotein following the protocol described by Madden and coworkers. [Cushing et al., Biochemistry, 47:10084-10098] This mixture was equilibrated for 20 min in the dark at room temperature. The unlabeled competitor peptide was dissolved and serially diluted in storage buffer supplemented with 5% DMSO (Sigma). Each serial dilution was aliquoted at 1/10 final volume, to which was added 9/10 volume of the protein/peptide mixture. All FP assays were performed in a 96-well format. Polarized fluorescence intensities were measured at 23 °C with a PerkinElmer Wallac Victor3 multilabel counter using excitation and emission wavelengths of 485 and 535 nm, respectively, for the FITC-labeled peptide. |
| Affinity data for this assay | |
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