| Assay Method Information | |
| | Binding to JNPL3 brain Hsp90 |
| Description: | The assay buffer (HFB) contained 20 mM HEPES (K) pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 0.01% NP40. Before each use, 0.1 mg/mL bovine gamma globulin (BGG) (Panvera Corporation, Madison, Wis.) and 2 mM DTT (Fisher Biotech, Fair Lawn, N.J.) were freshly added. GM-cy3B, a specific Hsp90 ligand, was synthesized as previously reported (10) and was dissolved in DMSO to form 10 μM solutions. Brains were homogenized in HFB with added protease and phosphatase inhibitors. Saturation curves were recorded in which GM-cy3B (3 nM) was treated with increasing amounts of brain homogenates. The Hill and Scatchard plot analyses of the experiment were constructed to show that at the low amounts of brain homogenates required to reach saturation, interaction from other cellular material was precluded. The amount of brain homogenate for which over 90% of GM-cy3B was Hsp90 bound at equilibrium (24 h) was chosen for the competition study. For the competition experiments, each 96-well contained 3 nM GM-cy3B, brain homogenate and tested inhibitor (initial stock in DMSO) in a final volume of 100 μL. The plate was left on a shaker at 4° C. for 24 h and the fluorescence polarization values in mP were recorded. EC50 values were determined as the competitor concentrations at which 50% of GM-cy3B was displaced. Fluorescence polarization measurements were performed on an Analyst GT instrument (Molecular Devices, Sunnyvale, Calif.). For GM-cy3B, an excitation filter at 545 nm and an emission filter at 610 to 675 nm were used with a dichroic mirror of 565 nm. Measurements were taken in black 96-well microtiter plates. |
| Affinity data for this assay | |
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