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 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.

Figure 3. IMAP FP generic phosphodiesterase assays
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.

Figure 4. IMAP TR-FRET generic phosphodiesterase assays
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 Progressive Binding System

IMAP Progressive Binding System lets researchers optimize their FP and TR-FRET assay conditions for each substrate used.  It consists of Progressive Binding Buffer A and Progressive Binding Buffer B, along with Progressive Binding Reagent.  The two Progressive Binding Buffers and the Progressive Binding Reagent can be combined in different proportions according to the acidic character of the substrate.

Benefits of the IMAP Progressive Binding System include

• Enables researchers to achieve maximum performance for every possible application when using IMAP.
• Can be used with virtually every kinase, phosphatase and phosphodiesterase
• Provides assay developers with the ability to easily address key variables such as substrate choice, desirable ATP concentrations and background parameters.

Table 1. Components of IMAP Progressive Binding System

IMAP Substrates

To facilitate the customization of the IMAP assay to fit specific needs, Molecular Devices offers a wide range of validated substrates and calibrators. These optimized IMAP substrates 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. These substrates may be used for the enzymes for which they were tested 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.

For more information, please see our IMAP Technology data sheet:

IMAP Technology datasheet

IMAP Assay Archive

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

Sample IMAP Assay Data

Figure 1: 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 2.  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.

IMAP^® Assays instrument compatibility

For more information on Molecular Devices' reagents and instrument compatibility, please see our Reagents Instrument Compatibility Chart.

Three options are available for purchasing the IMAP technology.

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).

R8166  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.

R8155  IMAP FP Evaluation Kit with Progressive Binding System

R8161 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.

R8175  IMAP FP PDE Evaluation Kits

R8176  IMAP TR-FRET PDE Evaluation Kits

• Includes optimized protocol, cAMP and cGMP substrates; customer provides their own PDE enzyme.

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.

R8073  IMAP FP Screening Express w/Original Binding System

R8127  IMAP FP Screening Express w/Progressive Binding System

R8160  IMAP TR-FRET Screening Express w/Progressive Binding System

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

R8062  IMAP FP Screening Express Kit Original Binding System

R8124  IMAP FP Screening Express Progressive Binding System

R8157  IMAP TR-FRET Screening Express Progressive Binding System

R8063  IMAP FP Bulk Kit Original Binding System

R8125  IMAP FP Bulk Kit Progressive Binding System with BSA

R8139  IMAP FP Bulk Kit Progressive Binding System with Tween

R8158  IMAP TR-FRET Bulk Kit Progressive Binding System with BSA

R8159  IMAP TR-FRET Bulk Kit Progressive Binding System with Tween

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