The IMAP® Substrate Finder Kits are assay development tools that accelerate the identification of suitable kinase substrates. The IMAP Substrate Finder enables researchers to test kinases against a variety of peptides, provided in a 384-well microplate, in order to rapidly identify potential substrates for subsequent IMAP screens. Additionally, substrate specificity may be evaluated among kinase variants or different mutants of the same kinase.
The Substrate Finder saves time and reduces costs. It allows users to test kinases against more than 50 different peptides in a single 384-well plate, for less than the cost of purchasing a handful of substrates separately. Once a substrate is identified, it can be used with IMAP in either FP or TR-FRET detection mode.
IMAP FP Substrate Finder Options
There are three IMAP FP Substrate Finder Kits: two for serine/threonine kinases and one fortyrosine kinases. All peptide substrates contained in these plates have been published and shown to be valid kinase substrates. All peptides are represented in quadruplicate so that replicates can be run, or multiple kinases can be tested for each substrate.
The kits provide coverage for different areas of the kinome:
- The first serine/threonine kinase plate covers the CAMK and AGC kinase groups. It contains 56 kinase substrates, plus three phosphorylated substrates as positive controls
- The second serine/threonine kinase plate covers the CMGC, CK1, STE, and TKL groups. It contains 61 kinase substrates, plus six phosphorylated control substrates
- The tyrosine kinase plate contains 57 kinase substrates and five phosphorylated control substrates
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. It provides 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.
The fluorescein- (5FAM-) labeled substrates are lyophilized in a 384-well microtiter plate.
Step 1: Upon reconstitution with 10 µL buffer, including ATP or other activators needed for the reaction, the peptides are at an appropriate concentration (100 nM) for an IMAP fluorescence polarization (FP) assay.
Step 2: Next, 10 µL of buffer with or without enzyme is added to the wells. Two of the replicate wells can be incubated with your kinase of interest, while the other two wells are used to assess the FP background specific for this particular peptide.
Step 3: The IMAP Binding System is added and the change in fluorescence polarization is measured. 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 plate, 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 one that initially had the highest ΔmP.
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 or 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 these systems:
- FlexStation 3 Benchtop Multi-Mode Microplate Reader
- SpectraMax M5e/M5 Multi-Mode Microplate Readers
- SpectraMax Paradigm Multi-Mode Microplate Reader
- SpectraMax i3 Multi-Mode Detection Platform (with Fluorescence Polarization Detection Cartridge – Fluorescein)
For more information, view the assay kits & instrument compatibility table.
IMAP FP Substrate Finder kits
Substrate Finder kit for Tyrosine kinases
Tyrosine plate consists of 62 substrates: 57 unphosphorylated substrates and 5 phosphorylated substrates (as controls)
Substrate Finder kit for Ser/Thr Kinases 1 (CAMK/AGC)
Serine/Threonine plate contains 59 substrates: 56 unphosphorylated substrates and 3 phosphorylated substrates (as controls)
Substrate Finder kit for Ser/Thr Kinases 2 (CMGC/CK1/ STE/TKL)
Serine/Threonine Kinase Plate 2 consists of 67 substrates: 61 kinase substrates and 6 phosphorylated substrates (as controls)
All IMAP FP Substrate Finder Kits include:
- Two 384-well plates with each substrate represented in quadruplicate
- 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 or visit our IMAP Assay Archive.