Plate reader reactions to assess NADase function were prepared as described previously4 (link). Reactions were built in 50 μl final volume with reaction buffer (20 mM HEPES-KOH pH 7.5, 100 mM KCl), 500 μM nicotinamide 1,N6-ethenoadenine dinucleotide; (ε-NAD, Sigma), 0.1–10 μM enzyme and 20 μM c-di-GMP. Reactions were prepared as master mixes in PCR-tube strips and initiated by adding nicotinamide 1,N6-ethenoadenine dinucleotide immediately before placing into the plate reader. Fluorescence emission at 410 nm was read continuously over 40 min using a Synergy H1 Hybrid Multi-Mode Reader (BioTek) after excitation at 300 nm. Plots were generated with GraphPad Prism 9.3.0.
Protocol detail
Fluorometric Assay for NADase Activity
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Corresponding Organization :
Other organizations : Harvard University, Dana-Farber Cancer Institute
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Variable analysis
independent variables
- Enzyme concentration (0.1–10 μM)
- Fluorescence emission at 410 nm
- Reaction buffer (20 mM HEPES-KOH pH 7.5, 100 mM KCl)
- Nicotinamide 1,N6-ethenoadenine dinucleotide (ε-NAD, 500 μM)
- C-di-GMP (20 μM)
- No positive or negative controls were explicitly mentioned.
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