Assay Method Information

Assay Name:  Fluorescence Polarization Assay
Description:  Fluorescence polarization (FP) data were measured on a microplate reader (Tecan Infinite M1000, Mannedorf, Switzerland) at 27° C. For Kd measurements, serially diluted protein in FP buffer (25 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT, and 0.02% (w/v) NaN3) supplemented with 0.1 mg/mL bovine IgG (Sigma) and 0.5 mM THESIT (Fluka) is dispensed into HE low-volume, black 96-well plates (Molecular Devices) and mixed with 3 nM of fluorescently labeled peptide, F*-STA02, pre-equilibrated with FP buffer in the dark for 10-15 minutes. Plates were mixed by vibration, centrifuged for 2 minutes at 1200 g to remove air bubbles and allowed to equilibrate for 30 minutes at room temperature before measurement. For competition binding experiments, protein at a concentration of 1-3×Kd was equilibrated with 3 nM fluorescently labeled reporter peptide for 30 minutes at room temperature. Unlabeled competitor peptides were serially diluted and mixed with the protein-reporter mix. Plates were mixed by vibration, centrifuged at 1200 g for 2 minutes and allowed to equilibrate at room temperature for 30 minutes before measurement.
Affinity data for this assay
 

If you find an error in this entry please send us an E-mail