IMAP FP Substrate Finder

An assay development tool that significantly reduces the largest barrier in kinase assay development: kinase substrate identification No antibodies Robust fluorescence signal Complete assay system Homogeneous assay Non-radioactive Sensitive detection Click to see Substrate Finder Assay Profiles
Protein kinases are one of the most important target classes in drug discovery today. The IMAP platform provides advanced tools for successful assay development and high-throughput screening (HTS) of kinases. The IMAP technology has a simple "mix-and-read" protocol: after the kinase reaction has been completed in a microtiter plate, the IMAP Binding Solution is added to stop the reaction and to enable detection by fluorescence polarization.

IMAP FP Substrate Finder

The IMAP FP Substrate Finder enables the researcher to quickly test one or more kinases against dozens of peptides included in a 384-well microtiter plate to identify potential substrates for subsequent IMAP screens. This new assay development tool significantly limits the time spent searching for a suitable substrate. Additionally, substrate specificity may be evaluated between different kinase variants or among different mutants of the same kinase. The Substrate Finder also allows the researcher to screen through dozens of predefined potential substrates for less than the cost of a few unknown potential 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.

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 (Figure 1) 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.

		Figure 1. IMAP Substrate Mapper plate layout for Ser/Thr CAMK and AGC kinases.

Simple Protocol

The fluorescein- (5FAM-) labeled substrates are lyophilized in a 384-well microtiter plate. (See Figure 2). Upon reconstitution with 10 µl buffer, including ATP or other activators needed for the reaction (Figure 2, 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. (See Figure 2, 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. (See Figure 2, Step 3). With four replicates of each substrate on a single plate, you may choose to assess several initial parameters against dozens of 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.

		Figure 2. Diagram of IMAP FP Substrate Finder assay principle.

Characterize your Kinase

The IMAP FP Substrate Finder can also help you characterize your kinase. In one set of experiments, the IMAP Substrate Finder revealed differences in substrate specificity between a wild-type and mutant form of a target kinase. This application could be easily configured to measure similar results in response to various lead compounds of interest. A novel kinase could be characterized according to its phosphorylation site preference in a very short amount of time. Alternatively, various enzyme preparations could be assayed for relative purity based on their phosphorylation patterns. These are just a few examples of how the IMAP Substrate Finder can enable new discoveries.

Sample Substrate Finder Results

Figure 3 shows the 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, MDC cannot guarantee that a substrate will be found for each kinase tested.

		Figure 3. IMAP FP Substrate Finder sample data.

Ordering Information

Go to the IMAP Ordering Information page.