AMP-Glo™ Assay

Description

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)

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