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| | RSK2 Inhibition Assay |
| Description: | RSK2 CTD and His6-ERK2 were expressed and purified as described (Cohen et al., Science, 308: 1318). The C436V mutant of RSK2 CTD was generated by Quikchange mutagenesis (Stratagene) and was indistinguishable from WT RSK2 CTD in kinase activity assays, as described previously. WT and C436V RSK2 CTD (10 uM) were activated by His6-ERK2 (10 uM) in 20 mM HEPES [pH 8.0], 10 mM MgCl2, 2.5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.2 mg/mL BSA and 200 uM ATP for 30 min at 22° C. Activated RSK2 CTD (5 nM) in 20 mM HEPES [pH 8.0], 10 mM MgCl2, 2.5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.25 mg/mL BSA, and 100 uM ATP were pre-incubated with inhibitors (ten concentrations, in duplicate) for 30 min. Kinase reactions were initiated by the addition of 5 uCi of [ -32P]ATP (6000 Ci/mmol, NEN) and 167 uM peptide substrate (RRQLFRGFSFVAK) (SEQ ID NO:1) and performed for 30 min at room temperature. Kinase activity was determined by spotting 5 uL of each reaction onto dried sheets of nitrocellulose that had been pre-washed with 1 M NaCl in 0.1% H3PO4. The nitrocellulose sheets were washed once with 1% AcOH solution and twice with a solution of 1 M NaCl in 0.1% H3PO4 (5-10 min per wash). Dried blots were exposed for 30 min to a storage phosphor screen and scanned by a Typhoon imager (GE Life Sciences). Data were quantified using the SPOT program (Knight, Z. et al. Nature Protocols, 2: 2459-66), and IC50 values were determined using GraphPad Prism 4.0 software. |
| Affinity data for this assay | |
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