| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | All fluorescence polarization experiments were conducted in 384-well, black, low volume, round-bottomed plates (Corning) using a BioTeck Synergy 2 plate reader (Winooski, VT). For binding experiments, to each well was added increasing amounts of protein and the 5-carboxyfluorescein-labeled Hsp70/90 C-terminal probe/tracer (20 nM). All wells had a final volume of 20 μl in the assay buffer (40 mM Tris-HCl, pH 7.4, 10% glycerol, 1 mM DTT). The plate was allowed to incubate at room temperature for 5 min toreach equilibrium. The polarization values in millipolarization units were measured at an excitation wavelength at 485 nm andan emission wavelength at 528 nm. An equilibrium binding isotherm was constructed by plotting the fluorescence polarization reading as a function of the protein concentration at a fixed concentration of tracer (20 nM). |
| Affinity data for this assay | |
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