| Solution Information | help | |
| Enzyme: | Ras-related C3 botulinum toxin substrate 1 | |
| inhibitor: | BDBM43266 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Bead sets are coated with individual GST-small G proteins, blocked with Buffer (0.01% NP-40; 30mM HEPES pH 7.5; 100mM KCl; 20mM NaCl; 1mM EDTA; 0.1% BSA and 1mM DTT), incubated overnight at 4 degrees C, and finally washed in buffer. The different bead sets, acquired from Duke Scientific, have similar size (~ 4 microm diameter) however they are distinguished by varied magnitude of emission at 665 +/-10 nm with excitation at 635 nm. The assay is conducted in 384-well microplates in a total well volume of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 200nM Bodipy-FL-GTP in buffer containing BSA and DTT for a final concentration of GTP of 100nM). Positive Controls, which contain bead mixture and fluorescent GTP but no test compound, are located in columns 1 and 2 on plate. Negative Controls containing bead mixture with fluorescent GTP and 0.5 mM unlabeled GTP, are collected from a separate test tube. Plates are placed on rotato | |
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