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| Solution Information | help | |
| Enzyme: | Heat shock 70 kDa protein 1A | |
| inhibitor: | BDBM31720 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | 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) Terbium-labeled anti-GST antibody (Tb-Ab) was purchased from Invitrogen. 4) Assay Buffer: 25 mM Bis-Tris, pH 7.0, 12.5 mM MgCl2, 1 mM DTT, 0.00625% Tween 20. 5) Hsp70/Fluorescein-ATP/Tb-Ab working solution contained 5 nM Hsp70, 12.5 nM fluorescein-12-ATP and 2.5 nM Tb-Ab in assay buffer. Solution was prepared fresh and kept on ice prior to use. Solution was incubated for 1 h at room temperature before dispensing into plates. 6) ADP working solution contained 25 uM ADP prepared fresh from 20 mM stock solution that was kept frozen. Hsp70 TR-FRET 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 | |
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