IMAP Evaluation Demo Kit

Kinase, Phosphatase, Phosphodiesterase Evaluation and Detection

Intended Use

The IMAP Evaluation Demo Kit allows one to set up calibration curves that can be used in conjunction with kinase, phosphatase and phosphodiesterase assays to determine percent phosphorylation in experimental samples. The calibration curves can also demonstrate microplate reader performance for FP and TR-FRET modes.

Principle of the Assay

IMAP technology is based on the specific, covalent-coordinate, high-affinity interaction of trivalent metal containing nanoparticles with phosphogroups. These phosphogroups can be free, or linked to serines, threonines, tyrosines, or other molecules, making 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 (FP) and time-resolved fluorescence resonance energy transfer (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 diagram.

		Figure 2. IMAP TR-FRET diagram. Components are the same as for FP, except that a Terbium (Tb) donor is added to the IMAP Binding System.

Kit Contents

• IMAP Progressive Binding Buffer A
• IMAP Progressive Binding Buffer B
• IMAP Reaction Buffer containing BSA
• IMAP Reaction Buffer containing Tween-20
• IMAP Progressive Binding Reagent
• Tb-Donor
• 5FAM-PKAtide
• 5FAM-PhosphoPKAtide

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

Assay Performance

		Figure 3. IMAP FP calibration curve with 5FAM-PKAtide and 5FAM-PhosphoPKAtide, read on SpectraMax M5e.

		Figure 4. IMAP TR-FRET calibration curves with 5FAM-PKAtide and 5FAM-PhosphoPKAtide, read on SpectraMax M5e. Blue circles, 0.3 µM peptide; red squares, 1.0 µM peptide; green triangles, 3.0 µM peptide.

Ordering Information

Go to the IMAP Ordering Information page.