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IMAP Assays

Homogeneous assays for accurate determination of kinase, phosphatase, and phosphodiesterase activities

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.

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

 IMAP FP generic kinase and phosphatase assays

IMAP FP assay principle
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

IMAP TR-FRET
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

IMAP PDE FP assay principle
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

IMAP PDE TR-FRET assay principle
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.

IMAP Assay Archive

For a complete profile of IMAP validated substrates with over 120 kinases, visit our IMAP Assay Archive.

Sample IMAP Assay data: ROCKII dilution curve

ROCKII dilution curve
Figure 1: 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.

FP vs. TR-FRET detection

IMAP data
Figure 2. 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.

 

IMAP Assays are compatible with the following systems:

Microplate Reader IMAP FP IMAP TR-FRET
SpectraMax i3 Microplate Reader X X
SpectraMax Paradigm Microplate Reader X X
SpectraMax M5e/M5 Microplate Readers X X
SpectraMax M4 Microplate Reader   X
FlexStation 3 Microplate Reader X X

For more information, view the assay kits & instrument compatibility table.

Three options are available for purchasing the IMAP kits.

OPTION 1: IMAP Evaluation Kits
These kits have been designed with a smaller volume for evaluation of the IMAP technology. They include beads and buffers for 800 data points in a standard 384-well format (20 mL enzyme reaction + 60 mL Binding Solution).

Description
Part Number
IMAP Evaluation Demo Kit
Optimized 800 data point IMAP kit which includes calibrators (phosphorylated and un-phosphorylated peptides) to run a calibration curve for either FP or TR-FRET detection
R8166
IMAP FP Evaluation Kit with Progressive Binding System
R8155
IMAP TR-FRET Evaluation Kit with Progressive Binding System
Optimized 800 data point protocol to run IMAP with the Progressive Binding System; Additional kinase and substrate are required
R8161
 IMAP FP PDE Evaluation Kits
R8175
 IMAP TR-FRET PDE Evaluation Kits 
Includes optimized protocol, cAMP and cGMP substrates; customer provides their own PDE enzyme
R8176


OPTION 2: IMAP Screening Express
IMAP Screening Express (also referred to as "Explorer") consists of the proprietary IMAP Binding Reagent and Binding Buffers for 8000 data points in a standard 384 well format. These kits are designed for customers who have their own enzyme and substrate, or purchase  substrates from our extensive validated substrate list.

Description Part Number
IMAP FP Screening Express w/Original Binding System R8073
IMAP FP Screening Express w/Progressive Binding System R8127
IMAP TR-FRET Screening Express w/Progressive Binding System R8160


OPTION 3: IMAP Purchase Plan (IPP) offers the best value for customers running multiple IMAP screens
IPP is designed for the customer performing several high-throughput screens in a year. This is the least expensive option for the IMAP Binding System. The Screening Express Progressive kits have both reaction buffers (Tween and BSA). For the Bulk kit, the customer can choose their preferred reaction buffer. The Original Binding System comes only with reaction buffer containing BSA as carrier. The Purchase Plan (R8064) consists of an annual subscription fee that allows the customer to buy any or all of the following.

Description
Part Number
IMAP FP Screening Express Kit Original Binding System 
R8062
IMAP FP Screening Express Progressive Binding System
R8124
IMAP TR-FRET Screening Express Progressive Binding System
R8157
IMAP FP Bulk Kit Original Binding System
R8063
IMAP FP Bulk Kit Progressive Binding System with BSA
R8125
IMAP FP Bulk Kit Progressive Binding System with Tween
R8139
IMAP TR-FRET Bulk Kit Progressive Binding System with BSA
R8158
IMAP TR-FRET Bulk Kit Progressive Binding System with Tween
R8159

Note: Any of the above purchases require subscription to IPP plan. Please contact your regional sales specialist for details.

For 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.