The fluorescein- (5FAM-) labeled substrates are lyophilized in a 384-well microtiter plate. (See figure above). Upon reconstitution with 10 µl buffer, including ATP or other activators needed for the reaction (Step 1), the peptides are at an appropriate concentration (100 nM) for an IMAP fluorescence polarization (FP) assay. Next, 10 µl of Buffer with or without enzyme is added into the plate. (Step 2). Two of the replicate wells can be incubated with your kinase of interest, while the other two wells contain background controls to assess the FP background specific for this particular peptide. The IMAP Binding System is added and the change in fluorescence polarization is measured. (Step 3). With four replicates of each substrate on a single plate, you may choose to assess several initial parameters against more than 50 different peptides in a single assay.

If you find more than one potential substrate with the IMAP FP Substrate Finder, we recommend evaluating your assay with the two or three substrates that have the highest ΔmP, i.e., the biggest change between no-enzyme control and enzyme. You may discover during the assay development process that one of the other substrates demonstrates an advantage over the initial one that had the highest FP on the IMAP FP Substrate Finder plate.

IMAP FP Substrate Finder Options

There are three IMAP FP Substrate Finder Kits: two focus on the Serine/Threonine kinases of the human kinome, and the third Substrate Finder concentrates on Tyrosine kinases. All peptide substrates contained in these plates have been published and shown to be valid kinase substrates (literature links are provided with the IMAP Substrate Mapper). The first Serine/Threonine plate covers the CAMK, AGC portions of the kinome and contains 56 kinase substrates and three phosphorylated substrates as positive controls. The second Serine/Threonine plate covers the CMGC, CK1, STE, TKL and contains 61 kinase substrates and six phosphorylated substrates as positive controls. The Tyrosine plate consists of 57 kinase substrates and five phosphorylated substrates. All peptides are represented in quadruplicate to allow for numerous controls or to enable testing of additional kinases, according to the user's discretion.

IMAP Substrate Mapper

The IMAP Substrate Mapper is an intuitive electronic map to assist in navigating the substantial amount of information that comes with the IMAP FP Substrate Finder: sequences of the peptide substrates, initial target kinase and location on the plate, literature references, recommended Progressive Binding Buffer ratios, and substrate part numbers to assist with ordering the desired substrate.

IMAP Assay Archive

For a complete profile of IMAP validated substrates with over 120 kinases, visit our IMAP Assay Archive.

IMAP Sample Assay Data

Results from a standard screen for a kinase substrate.

The Ser/Thr Kinase RSK-1 was assayed at 0.05 U/ml and 100 µM ATP. Eight substrates were identified that demonstrated a polarization change ΔmP (plus enzyme - background) of > 100 mP, one of which had a ΔmP of > 300 mP.

Note: While each of the peptides in the Substrate Finder was chosen with great care, Molecular Devices cannot guarantee that a substrate will be found for each kinase tested.

IMAP Substrate Finder Kits are compatible with the following Molecular Devices microplate readers:

• FlexStation® 3 Microplate Reader
• SpectraMax® M5e Microplate Reader
• SpectraMax® M5 Microplate Reader
• SpectraMax® M4 Microplate Reader

For more information on Molecular Devices' reagents and instrument compatibility, please see our Reagents Instrument Compatibility Chart.

IMAP FP Substrate Finder Kits

All IMAP FP Substrate Finder Kits include:

• Two 384-well plates. Each substrate is represented in quadruplicate on each plate.
• Sufficient IMAP beads and buffers to run both plates, including added controls.
• IMAP Substrate Mapper (electronic file).
• IMAP Substrate Finder Product Insert (contains directions and a sample protocol).

Validated substrates for IMAP Assays

For more information on substrates available for purchase, including amino acid sequences and part numbers, please contact your Molecular Devices regional sales specialist.