| Solution Information | help | |
| Enzyme: | Heat shock 70 kDa protein 1A | |
| inhibitor: | BDBM31758 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Hsp70 fluorescence polarization assay protocol. Hsp70 assay materials: 1)Hsp70 protein (GST-tagged 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)Hsp70/Fluorescein-ATP working solution contained 12.5 nM Hsp70 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 10 uM ADP prepared fresh from 20 mM stock solution that was kept frozen. Hsp70 dose response confirmation protocol: 1)Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well | |
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