IMAP Kinase, Phosphatase, and Phosphodiesterase Assays
Homogeneous assay for accurate determination of kinase, phosphatase, and phosphodiesterase activities
IMAP® technology provides a homogeneous assay applicable to a wide variety of kinases without regard for substrate peptide sequence. The assay is a simple "mix-and-read" procedure that allows accurate determination of kinase, phosphatase, and phosphodiesterase (PDE) activity.
Based on the specific, high-affinity interaction of trivalent metal-containing nanoparticles with phosphogroups, IMAP is a generic platform to assess kinase, phosphatase, and phosphodiesterase activity. An enzyme reaction is performed using fluorescently labeled substrate. Addition of the IMAP Binding System stops the enzyme reaction and specifically binds the phosphorylated substrates. Binding of the substrate to the beads can be detected using either FP or TR-FRET as a readout.
Benefits of IMAP Assays
- IMAP provides a complete assay system for screening kinases, phosphatases, and phosphodiesterases.
- Because IMAP assays are not antibody-based, they are generic and can be used for any kinase, phosphatase, or phosphodiesterase.
- Robust fluorescence signal gives reliable results with good Z factors.
- IMAP assays are homogeneous and amenable to miniaturization for greater cost savings.
- IMAP assays are available in both FP and TR-FRET detection modes to meet users' screening needs.
IMAP Progressive Binding System
IMAP Progressive Binding System lets researchers optimize their FP and TR-FRET assay conditions for each substrate used. It enables researchers to achieve maximum performance for every possible application when using IMAP. The system consists of Progressive Binding Buffer A and Progressive Binding Buffer B, along with Progressive Binding Reagent. The two buffers and the reagent can be combined in different proportions according to the acidic character of the substrate choice, desirable ATP concentrations and background parameters.
Table 1. Components of IMAP Progressive Binding System
|IMAP Progressive Binding Buffer A||Baseline Binding Buffer|
|IMAP Progressive Binding Buffer B||Affects TR-FRET background by reducing, or "blocking", the non-phosphate-based binding of the fluorescent substrate to the Binding Reagent|
|IMAP Progressive Binding Reagent
||Introduces the phosphate binding entities. This Binding Reagent specifically binds to phosphate residues via a coordinate covalent complex bond.|
|IMAP TR-FRET Donor
(Tb based Phospho conjugate)
|Enables the TR-FRET read-out based on the close proximity of Tb-Donor and fluorophore on phosphorylated substrate by binding both even under very stringent Binding Buffer conditions to the Binding entities|
To facilitate the customization of the IMAP assay, we offer a wide range of validated substrates and calibrators. These are offered individually, as a complement to the IMAP Screening Express and IMAP Purchase Plan (IPP). The IMAP Screening Express and IPP programs allow greater flexibility by providing the detection portion of the IMAP assay, leaving the enzymatic reaction to be determined by the researcher. Substrates may be used for the enzymes for which they were originally identified or as potential substrates for alternate enzymes.
- The binding solution conditions for the validated IMAP substrates are optimized to ensure the finest performance when using the IMAP platform.
- Substrates come in two sizes and can be used with both IMAP FP and IMAP TR-FRET.
- To date, we have validated dozens of different substrate sequences with over 100 kinases, and additional calibrator peptides.
- Many substrates are available with both red and green labels, enabling multiplexing and providing a way to address issues with compound autofluorescence interference, present in some compound libraries.