| Assay Method Information | |
| | PKC AlphaScreen Assay |
| Description: | Interaction assays were performed using the Alpha-Screen (amplified luminescent proximity homogeneous assay) technology, as described previously [Lopez-Garcia et al., Chem. Biol., 18:1463-1473; Zhang et al., Chem. Biol., 21:754-765]. The bead-based assay allows the detection of biomolecular interactions and the effect of small compounds. Briefly, the Alpha-Screen assay was performed in a finalvolume of 25 μL in white 384-well microtiter plates (Greiner Bio-One) with His-PKCι-CD (40 nM) and biotin-pseudosubstrate (80 nM) or His-PKCι-CD (15 nM) and biotin-ROCKtide (100 nM) in a buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.01% (v/v) Tween 20, 0.1% (w/v) BSA, 2 mM dithiothreitol (for the PKCι-Rocktide interaction) or 5 mM dithiothreitol (for the PKCι-pseudosubstrate interaction), and the corresponding concentration of the compound. Five microliters of beads at a concentration of 20 μg/mL was added to the mixture (nickel chelate-coated acceptor beads and streptavidin-coated donor beads), and after incubation for 90 min, Alpha-Screen counts were obtained in an EnVision Multiplate reader. |
| Affinity data for this assay | |
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