| Assay Method Information | |
| | AlphaScreen Assay |
| Description: | The AlphaScreen Assay (Perkin Elmer) was performed according to the manufacturer's protocol in 384-well microtiter plates. Incubations were performed in white 384-well microtiter plates containing 20uL of a reaction mix (50mM Tris-HCl pH 7.4, 100mM NaCl, 1mM dithiotreitol, 0.01% (v/v) Tween-20, 0.1% (w/v) BSA, 1mM DMSO, 100nM His-CaPkh2 or 100nM His-PDK1, 50nM Biotin-PIFtide, and the indicated concentrations of unlabeled PIF-tide or small compounds) and 5uL of beads. 5uL of beads solution containing nickel chelate-coated acceptor beads and streptavidin-coated donor beads (20ug/mL final concentrations) was added to the reaction mix. The reaction mix and the beads were incubated in the dark for 45 min at RT and the emission of light from the acceptor beads was measured in the EnVision reader (Perkin Elmer) and analyzed using the GraphPad Prism software. |
| Affinity data for this assay | |
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