IMAP FP & TR-FRET Phosphodiesterase (PDE) Evaluation Assay Kits

Rapid, Sensitive, Homogeneous, Non-Radioactive Assay for Phosphodiesterases

• Direct measurement of AMP (adenosine monophosphate) or GMP (guanosine monophosphate)
• Easy mix and read homogeneous protocol
• Generic platform
• No antibodies
• No radioactivity
• Two detection options – FP and TR-FRET
• Robust, reproducible
• 96, 384, 1536 format

Phosphodiesterases, a superfamily of enzymes involved in regulating intracellular signaling, are emerging as a promising class of drug targets. Screening technologies that provide direct, rapid and reproducible results are critical to researchers in their search for potential PDE inhibitors.

IMAP is well known for its use in kinase screening due to its homogeneous, antibody-free detection of phosphorylation. This technology is also excellent for the evaluation of phosphodiesterases, as well as phosphatases. This assay is a simple, homogeneous “mix and read” procedure that allows accurate determination of enzyme activity.

Intended Use

The IMAP PDE FP; TR-FRET Evaluation kits are designed for use in biochemical in-vitro assays of phosphodiesterase activity. Kits provide both Fluorescein labeled cAMP and cGMP as possible substrates to evaluate the optimal configuration for the assay. The kits provide a protocol to run the assay with IMAP components for 800 data points. All that is needed is the PDE of choice.

Principle of the Assay

The IMAP technology is based on the high affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. This IMAP “Binding Reagent” complexes with phosphate groups on phosphopeptides generated in a kinase reaction or on nucleotide monophosphate generated from cyclic nucleotides (cAMP/cGMP) through phosphodiesterases. With FP detection 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 1. Principle of the IMAP FP PDE assay system

With TR-FRET detection 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.

		Figure 2. Principle of the IMAP TR-FRET PDE assay system

Kit Contents

IMAP FP PDE Progressive Binding System	IMAP TR-FRET PDE Progressive Binding System
IMAP Progressive Binding Buffer A	IMAP Progressive Binding Buffer A
IMAP Progressive Binding Buffer B	IMAP Progressive Binding Buffer B
IMAP Progressive Binding Reagent	IMAP Progressive Binding Reagent
IMAP Reaction Buffer containing BSA	IMAP Reaction Buffer containing BSA
IMAP Reaction Buffer containing Tween-20	IMAP Reaction Buffer containing Tween-20
Fluorescein- labeled cAMP* Substrate	Fluorescein- labeled cAMP* Substrate
Fluorescein- labeled cGMP* Substrate	Fluorescein- labeled cGMP* Substrate
	IMAP TR-FRET Donor (=Tb based Phospho conjugate)

When used as suggested, each kit provides sufficient reagents for 800 data points in 384-well plate format.

Assay Performance

		Figure 3. Enzyme dilution curves using IMAP-FP-detection (A) or IMAP-TR-FRET (B) for PDE5 (Calbiochem) with 100 nM Fam cGMP and PDE4A4 (Calbiochem) with 100 nM Fam-cAMP as substrates in IMAP Reaction Buffer (Tween-20) using the IMAP Progressive Binding System.
		Figure 3A Figure 3B

Ordering Information

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