IMAP FP Substrate Finder

The IMAP® Substrate Finder Kits are assay development tools that can (a) accelerate the identification of substrates for kinases and (b) be used to profile substrate specificity of a kinase.  The IMAP Substrate Finder enables researchers to test kinases against a variety of peptides included in a 384-well microtiter plate to rapidly identify potential substrates for subsequent IMAP screens.  Additionally, substrate specificity may be evaluated between kinase variants or among different mutants of the same kinase. The Substrate Finder also allows users to screen through many predefined potential substrates for less than the cost of a few substrates. Once a substrate is identified, it can be used with IMAP in either FP or TR-FRET detection mode. Depending on the mode chosen, optimized conditions may vary.

Benefits of IMAP Substrate Finder

·               Save time by testing kinases against more than 50 different peptides in a single 384-well plate.

·               Screen dozens of predefined potential substrates for less than the cost of a few substrates.

·               Expand screening options by using identified substrates with IMAP in either FP or TR-FRET detection mode.

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 users 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 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.