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Setting up a kinase profiler with IMAP

olution, and then reads the FP. Wells without enzyme serve as negative controls for each kinase assay, and calibrator phosphopeptides provide the positive controls. The plates can be read by any instrument capable of FP; the MDC Analyst and SpectraMax M5 instruments are recommended. For the fluorescein-labeled substrates, we use a filter set of 485 nm (excitation), 505 nm (dichroic), and 530 nm (emission) to measure the FP. Analysis and navigation tools are included with the IMAP Substrate Finder kits for fast and easy determination of assay results.

The histogram in Figure 2 shows the initial SF Ser/Thr 1 assay for AMPK, the first kinase on the Table 1 panel for which a substrate for IMAP has not yet been determined. Please refer to www. moleculardevices.com for a more detailed Substrate Finder assay protocol. The concentration of kinase is expressed in U/mL for all data in this note, where 1 U = 1 nmol phosphate incorporated into a substrate per minute.

Several considerations may influence the kinase concentration chosen for Substrate Finder experiments. 1.0 U/mL of AMPK was selected because it would provide sufficient enzyme to pick up substrate hits but still enable a significant dilution of the stock in the assay. In some of the subsequent Substrate Finder assays presented in this note, we chose to use a lower concentration of kinase, either because our stock of enzyme was limiting, or we had prior knowledge of the sensitivity of at least one of the substrates in the assay plate. In a few cases, it may be necessary to repeat a Substrate Finder assay using a different kinase concentration in order to obtain useful information. In most of the work described in
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