Protein Kinase Assay Development using Caliper LabChip® Systems
Aileen Zhou, Theo Nikiforov, Hugh Daniels, Rex Hardy, Wally Parce and Peter Panfili
Caliper Technologies Corp. Mountain View, CA
Summary:
We report a new and direct assay for protein kinase activity using Caliper Technologies Corp. LabChip® microfluidics systems. The kinase phosphorylates the substrate, producing a change in charge and hence a shift in electrophoretic mobility. Because the substrate and product contain a fluorescent label, the detection of a shift in mobility is rapid, sensitive and direct. No antibody is required and as little as nanoliter-scale fluid is needed in the assay. Assay development is fast and relatively simple.
Enzymes for which fluorogenic substrates are not available can be conveniently assayed on microfluidic chips in one of two formats: the on-chip and off-chip mobility-shift assay. In the on-chip version of the assay, solutions of the enzyme and substrate are placed in separate wells of the microchip, and the compounds to be evaluated are sipped from the microtitration plate via a capillary. Upon application of a constant vacuum and an electric field, materials are continuously mixed and product separated in the main reaction channel. Consumption of target and substrate is exceedingly low. The off-chip mobility shift assay is a continuous flow format where enzyme, substrate and compound are mixed and incubated in the microtitration plate. Small volumes of the reaction mixtures are introduced via the attached capillary sipper by the continuous application of vacuum to one of the wells of the chip. Substrate and product are then separated by the application of a high electric field and detected using fluorescence. This is suitable for enzymes with low enzymatic rates.
Debbie Moon