IMAP Assays Substrate Finder
The IMAP FP Substrate Finder enables researchers to quickly test one or more kinases against dozens of peptides to identify potential substrates for subsequent IMAP screens.
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.