Enzyme – IMAP Assays

IMAP® technology provides a homogeneous assay applicable to a wide variety of kinases, phosphatases, and phosphodiesterases without regard for substrate peptide sequence. The assay is a simple mix-and-read procedure allowing accurate determination of enzyme activity.


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Based on the specific, high-affinity interaction of phospho groups with trivalent metal-containing nanoparticles (beads), IMAP is a generic, non-antibody-based 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 initiates binding of the beads to phosphorylated substrates. Binding of the substrate to the beads, which correlates to enzyme activity, 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. 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.

Components of IMAP Progressive Binding System

Reagent Description
IMAP Progressive Binding
Buffer A
Baseline binding buffer
IMAP Progressive Binding
Buffer B
Affects 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
[Terbium (Tb) based phospho conjugate]
Enables the TR-FRET read-out by binding to fluorophore on phosphorylated substrate via the attached phosphate binding entities

IMAP Substrates

Molecular Devices offers a wide range of validated substrates and calibrators. Substrates may be used with the enzymes for which they were originally validated, or as potential substrates for other enzymes.

  • Validated IMAP substrates with optimized binding conditions ensure the best performance when using the IMAP platform
  • Substrates come in two sizes and can be used with both IMAP FP and IMAP TR-FRET
  • Dozens of different substrates have been validated with over 100 enzymes. Many substrates are available with either red or green fluorescent label, enabling multiplexing and providing a way to address issues arising from compound autofluorescence

Figure 1: Calibration curve, 96-well assay format. Calibration curve for the CatchPoint cyclic-GMP Kit. Data was taken 60 minutes after addition of Stoplight Red substrate. The EC50 of the cGMP calibration curve was 2.2 nM (264 fmol). Minimum detectable concentration was 0.2 nM (24 fmol).


Figure 2: Atrial natriuretic peptide dose response in RFL-6 cells, 96-well assay format. A confluent monolayer of RFL-6 cells in a 12-well plate was pre-incubated for 10 minutes in Krebs Ringer bicarbonate buffer containing the non-selective phosphodiesterase inhibitor IBMX. Cells were subsequently stimulated with ANP for 15 minutes, lysed, and analyzed using the cGMP Fluorescent Assay Kit.

IMAP FP generic kinase and phosphatase assays


Figure 1. IMAP principle using FP readout: Binding Solution is added after the kinase reaction using a fluorescently labeled peptide. The small phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its polarization.

IMAP TR-FRET generic kinase and phosphatase assays


Figure 2. IMAP principle using TR-FRET readout: Binding Solution is added after the kinase reaction using a fluorescently labeled peptide or protein. In this system, the nanoparticle is spiked with a Terbium (Tb)-Donor molecule. By binding to the spiked M(III)-based nanoparticles, the phosphorylated fluorescent substrate comes into close proximity with the Tb-Donor, which allows measurement of the TR-FRET between the Tb-Donor and the phosphorylated, fluorescent substrate.

IMAP FP generic phosphodiesterase assays


Figure 3. IMAP principle using FP readout: Binding Solution is added after the phosphodiesterase reaction using a fluorescently labeled substrate. The small phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its polarization.

IMAP TR-FRET generic phosphodiesterase assays


Figure 4. IMAP principle using TR-FRET readout: Binding Solution is added after the phosphodiesterase reaction using a fluorescently labeled substrate. In this system, the nanoparticle is spiked with a Terbium (Tb)-Donor molecule. By binding to the spiked M(III)-based nanoparticles, the phosphorylated fluorescent substrate comes into close proximity with the Tb-Donor, which allows measurement of the TR-FRET between the Tb-Donor and the phosphorylated, fluorescent substrate.