| Assay Method Information | |
| | In Vitro Inhibition Assay |
| Description: | The reagents used have the following composition: Enzyme buffer (EB): 50 mM HEPES (pH: 7.0) (Sigma H7523), 100 mM NaCl (Sigma S7653), NaN.sub.3 at 0.01% (Sigma S8032), BSA at 0.005% (Sigma A2153), 0.05 mM sodium orthovanadate (Calbiochem 567540). Detection buffer (DB): 50 mM HEPES (pH: 7.0), BSA at 0.1%, 0.8 M KF (Fluka 60239), 20 mM EDTA (Sigma E5134).The peptide used is the one described in Biochemistry, 2005, 44, 8533-8542; A-21-K(biotin)NH.sub.2, obtained from NeoMPS (reference SP081233). All the HTRF reagents Mab PT66-K (61T66KLB) and streptavidin-XL665 (610SAXLB), and the SEB reagent, are purchased from Cisbio.The test is carried out in a 384-well plate (Greiner 784076). The serial dilutions are carried out in pure DMSO, and then an intermediate one-in-three dilution in water is carried out, with 1 microliter of each concentration being distributed, all these operations being performed using the Zephyr apparatus (Caliper Life Sciences). |
| Affinity data for this assay | |
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