In the assay, the amount of AMP produced by the reaction is proportional to the light measured, which can be extrapolated using a standard curve (Figure 2A). Reactions that use ATP as a substrate to produce AMP, such as ubiquitin ligases, require the removal of residual ATP before converting AMP to ATP; reactions that do not use ATP as a substrate, such as cAMP-dependent phosphodiesterases, do not require this step. Thus, enzymatic reactions of the latter type can be combined with the step of converting AMP to ADP.
Figure 2A shows the standard curves generated for up to 10µM AMP simulating enzymatic reactions generating different amounts of AMP, with or without ATP as a substrate. The signal to background ratios (SB) resulting from the AMP concentration are also shown. This assay is capable of detecting low AMP production, to less than 30nM AMP without ATP and approximately 150nM with ATP present. The AMP-Glo Assay is sensitive to low AMP concentrations and the luminescent signal, reported as RLUs, is linear with increasing AMP amount.
The assay is robust as indicated by the Z’ value (greater than 0.8) and is also less susceptible to false hits (Figure 2B). During a screen of 1,280 compounds of the LOPAC library performed using the AMP-Glo reagents in the presence of AMP, only three compounds altered luminescence by more than 80% indicating that the AMP-Glo Assay is resistant to chemical interference and is highly suitable for HTS.