| Assay Method Information | |
| | PKC Enzyme Assay |
| Description: | Protein kinase C (PKC), zeta activity was measured by following the phosphorylation of a biotinylated peptide substrate in the presence of ATP using AlphaScreen technology (Perkin Elmer, Waltham, MA). The AlphaScreen assay format relies on bringing a streptavidin coated donor bead and an IgG-protein A acceptor bead into proximity through the interaction between biotinylated phosphorylated peptide and a phospho-specific antibody to the peptide. Upon excitation of the donor bead at 680 nm, excited state singlet oxygen is produced and diffuses to acceptor beads in close proximity (200 nm), activating a cascade of fluorophores resulting in emission between 520-620 nm. Kinase activity is quantified through the evaluation of this emission signal. Compounds are evaluated as potential inhibitors of the PKC kinase activity by measuring their effects on the phosphorylation of the peptide substrate. IC50 value determinations were made for potential inhibitors by testing compounds at 12 concentrations in three-fold serial dilutions with each concentration tested in triplicate. Reactions were carried out for 60 minutes at room temperature. Reactions were then quenched by addition of stop solution. Stop solution included AlphaScreen acceptor and donor beads as well as phospho-specific antibody #2261(Cell Signaling). Plates were then read on a Fusion reader after an overnight equilibrium at room temperature. |
| Affinity data for this assay | |
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