Introduction to IMAP

Complete assay system for screening kinases, phosphatases, and phosphodiesterases No antibodies Robust fluorescence signal Complete assay system Homogeneous assay Non-radioactive Sensitive FP and TR-FRET detection Explore IMAP Explore our exclusive IMAP technology using the links in the left menu.

Until now, assays of kinase activity have been performed using radioactive isotopes or highly specific antibodies. To address this, Molecular Devices introduced its proprietary IMAP® technology, providing a non-radioactive, homogeneous assay applicable to a wide variety of kinases without regard for the substrate peptide sequences. The assay is a simple "mix-and-read" procedure that allows accurate determination of enzyme activity.

What is IMAP?

IMAP is a technology based on the specific, covalent-coordinate, high-affinity interaction of trivalent metal containing nanoparticles with phosphogroups. These phosphogroups can be free, linked to serines, threonines or tyrosines, or other molecules which make IMAP a generic platform to assess kinase, phosphatase and phosphodiesterase activity. This basic principle has been used in the IMAP Binding System using both fluorescence polarization and TR-FRET (as a read-out). In a microwell assay format, fluorescently-labeled peptides are phosphorylated in a kinase reaction. Addition of the IMAP Binding System stops the kinase reaction and specifically binds the phosphorylated substrates. Phosphorylation and subsequent binding of the substrate to the beads can be detected either by FP (Figure 1) or TR-FRET (Figure 2).

		Figure 1.  IMAP FP generic kinase and phosphatase assays 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.

		Figure 2.  IMAP TR-FRET generic kinase and phosphatase assays 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 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.

		Figure 3.   IMAP FP generic phosphodiesterase assays IMAP principle using FP readout: The IMAP “Binding Reagent” complexes on nucleotide monophosphate generated from cyclic nucleotides (cAMP/cGMP) through phosphodiesterases.  Such binding causes a change in the rate of the molecular motion of the phosphate bearing molecule, and results in an increase in the fluorescence polarization value observed for the fluorescent label attached to the substrate.

		Figure 4.  IMAP TR-FRET generic phosphodiesterase assays IMAP principle using TR-FRET: The IMAP “Binding Reagent” complexes on a nucleotide monophosphate generated from cyclic nucleotides (cAMP/cGMP) through phosphodiesterases.  Such binding causes the fluorophor on the Product to come in close proximity to the Tb-Donor also binding to the Binding entities and Fluorescence Resonance Energy Transfer (FRET) is generated upon excitation. Due to the long lifetime of the Tb-Donor this can be measured in a Time-resolved mode, which further reduces the Background of the assay.

Strong Signals

Assays using IMAP technology deliver an intense fluorescent signal that produces high precision and robust results, even in the presence of interfering compounds.

Simple Protocol

The IMAP Kinase and PDE Assay protocols are easy to use. The kinase or PDE reaction mixture is incubated with the binding reagent and the microplate is then read by an Analyst® GT or SpectraMax® M5 multimode instrument from Molecular Devices.

		Figure 5.  ROCKII dilution curve Enzyme dilution curves for ROCKII in BSA and in Tween IMAP Reaction Buffer using the Progressive Binding System. Reaction conditions: ROCKII kinase (Upstate: 14-451) as indicated, 100 nM FAM-S6 derived substrate (5FAM-AKRRRLSSLRA-COOH, R7184), 100 µM ATP. IMAP Binding Solution conditions: 100% Binding Buffer A, Progressive Binding Reagent 1:400.

		Figure 6.  FP vs. TR-FRET detection Akt inhibition with staurosporine: Comparison of IMAP TR-FRET (top) and IMAP FP (bottom) detection. Reaction conditions: Akt kinase (0.1u/ml, Upstate: 14-276), 300 nM, 1µM, 3µM FAM-Crosstide (5FAM-GRPRTSSFAEG-COOH, R7110), 10 µM ATP, Staurosporine as indicated. IMAP Binding Solution: 40% Binding Buffer A, 60% Binding Buffer B, Progressive Binding Reagent 1:400.

		Figure 7.  PKA dilution curve PKAtide substrate dilution curves with PKA using IMAP TR-FRET. Reaction conditions: PKA kinase at 0.01, 0.003, 0.001 U/ml (Upstate: 14-440), FAM-PKAtide as indicated (5FAM-GRTGRRNSI-NH2, R7250), 100 µM ATP, IMAP Binding Solution: 95% Binding Buffer A, 5% Binding Buffer B, Progressive Binding Reagent 1:400.

		Figure 8. Enzyme dilution curves using IMAP-FP for PDE5 (Calbiochem) with 100 nM Fam cGMP and PDE4C2 (Calbiochem) with 100 nM Fl-cAMP as substrates in IMAP Reaction Buffer (Tween-20) using IMAP Progressive Binding System.

		Figure 9. Enzyme dilution curves using IMAP-TR-FRET for PDE5 (Calbiochem) with 100 nM Fl-cGMP and PDE4C2 (Calbiochem) with 100 nM Fl-cAMP as substrates in IMAP Reaction Buffer (Tween-20) using IMAP Progressive Binding System.