AMP-Glo™ Assay

The AMP-Glo™ Assay is a homogeneous assay that generates a luminescent signal from any biochemical reaction that produces AMP as a reaction product. This versatile system can measure the activity of a broad range of enzymes, such as cyclic AMP-specific phosphodiesterases, aminoacyl-tRNA synthetases, DNA ligases and ubiquitin ligases or enzymes modulated by AMP. The AMP-Glo™ Assay is designed to quantitatively monitor the concentration of AMP in a biochemical reaction in a wide range of plate formats, including high-throughput formats. The stable luminescent signal of the assay eliminates the need...

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The AMP-Glo™ Assay is a homogeneous assay that generates a luminescent signal from any biochemical reaction that produces AMP as a reaction product. This versatile system can measure the activity of a broad range of enzymes, such as cyclic AMP-specific phosphodiesterases, aminoacyl-tRNA synthetases, DNA ligases and ubiquitin ligases or enzymes modulated by AMP. The AMP-Glo™ Assay is designed to quantitatively monitor the concentration of AMP in a biochemical reaction in a wide range of plate formats, including high-throughput formats. The stable luminescent signal of the assay eliminates the need for an injector-equipped luminometer and allows batch-mode processing of multiple plates. The assay can be used to determine the AMP produced either in the presence or absence of ATP as a substrate.

The assay contains two reagents: one to terminate the AMP-generating enzymatic reaction and simultaneously remove ATP and convert AMP produced into ADP, and a second reagent that converts the ADP to ATP followed by conversion of the ATP into a luminescent signal using the luciferin/luciferase reaction. The assay also is well suited for monitoring AMP produced in biochemical reactions catalyzed by enzymes that do not use ATP as a substrate, such as cAMP-dependent phosphodiesterases (PDE) and bacterial DNA ligases.

The AMP-Glo™ Assay has a high dynamic range and produces a strong signal at low substrate conversion, making it well suited for screening low activity enzymes. The assay produces minimal false hits and Z´ values greater than 0.7.

Features - Benefits

• High Signal Strength at Low Substrate Conversion: Measure enzyme activity that more closely mimics physiological conditions—very well suited for low-activity enzymes.
• Sensitive to Low Concentrations of AMP: Requires less enzyme than other assays; cost savings.
• Universal: Use the assay with virtually with any AMP-producing enzyme—enables screening of a wider range of enzymes using a single platform.
• Accurately Measures AMP Levels at a Wide Range of Starting Substrate Concentrations: Activity measured truly reflects enzyme activity and is well suited for measuring the effects of inhibitor on enzyme activity.
• Luminescent Readout: Much less susceptible to interference from library compounds than fluorescent-based methods.

Applications

• Screen library compounds for effects on target enzymes.
• Use with a wide range of enzymes:
  • ubiquitin ligases
  • cyclic AMP-specific phosphodiesterases
  • aminoacyl-tRNA synthetases
  • DNA ligases
  • poly(A) deadenylases
  • demethylases (indirect method)

Notes

The AMP-Glo™ Assays contain enough reagent to perform the indicated number of reactions in a low-volume 384-well plate format using 5μl of reaction enzyme, 5μl of AMP-Glo™ I Reagent and 10μl of AMP Detection Solution. The system also can be used in standard 96-well plates (25μl:25μl:50μl).

References

1. Zhang, L. et al. (2008) Cyclic nucleotide phosphodiesterase profiling reveals increased expression of phosphodiesterase 7B in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 105, 19531–6.
2. Guo, M. et al. (2010) New functions of aminoacyl-tRNA synthetases beyond translation. Nature Rev. Mol. Cell. Biol. 11, 668–74.
3. Mills, S.D. et al. (2011) Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo. Antimicrob. Agents Chemother. 55, 1088–96.
4. Robinson, P.A. and Ardley, H.C. (2011) Ubiquitin-protein ligases. J. Cell Sci. 117, 5191–4.
5. Galperin, M.Y. and Koonin, E.V. (1997) A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity. Protein Science 6, 2639–43.
6. Steinberg, G.R. and Kemp, B.E. (2009) AMPK in health and disease. Phyisol. Rev. 89, 1025–78.

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