| Solution Information | help | |
| Enzyme: | Heat shock cognate 71 kDa protein | |
| inhibitor: | BDBM31701 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Hsc70 fluorescence polarization assay protocol. Hsc70 assay materials: 1) Hsc70 protein (ATPase domain) was provided by Prof. John Reed laboratory (Sanford-Burnham Medical Research Institute, San Diego, CA). 2) Fluorescein-12-ATP (catalogue NEL439001EA) was purchased from PerkinElmer. 3) Assay Buffer: 25 mM Bis-Tris, pH 7.0, 12.5 mM MgCl2, 1 mM DTT, 0.00625% Tween 20. 4) Hsc70/Fluorescein-ATP working solution contained 81.25 nM Hsc70 and 12.5 nM fluorescein-12-ATP in assay buffer. Solution was prepared fresh and kept on ice prior to use. 5) ADP working solution contained 25 uM ADP prepared fresh from 20 mM stock solution that was kept frozen. Hsc70 HTS protocol: 1) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 1-22 of Greiner 384-well black small-volume plates (784076). 2) Column 23 was utilized for negative control samples and added with 4 uL of 10% DMSO using WellMate bulk dispenser (Matrix). 3) Column 24 was utilized for positive control samples and added wit | |
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