96 well half area black flat bottom polystyrene nbs microplates

^CgluPup-Q30C was labeled with fluorescein-5-maleimide as described before [13 (link)]. To generate ^CgluPup-Q30C-Fluorescein ~ ^MtbFabD and ^CgluPup-Q30C-Fluorescein ~ ^MtbPanB, 10 μM of ^CgluPup-Q30C-Fluorescein was incubated with 20 μM ^MtbFabD or ^MtbPanB in the presence of 10 mM ATP and 2 μM ^CgluPafA-His₆ in a total reaction volume of 200 μl for 5 h at room temperature. ^CgluPafA-His₆ was removed as before using a Ni-NTA spin column and the flow-through was concentrated using an Amicon spin concentrator with molecular weight cutoff of 30 kDa. The concentration was determined by measuring absorbance at 492 nm and using an extinction coefficient of 83,000 M⁻¹cm⁻¹ (4 μM ^CgluPup-Q30C-Fluorescein ~ ^MtbFabD and 5.5 μM ^CgluPup-Q30C-Fluorescein ~ ^MtbPanB). All anisotropy measurements were performed on a Synergy BioTek plate reader using Corning® 96 Well Half Area Black Flat Bottom Polystyrene NBS™ Microplates (product code 3993). 12.5 nM of the ^CgluPup-Q30C-Fluorescein labeled substrate or free ^CgluPup was incubated with increasing concentrations of ^CgluPafA (0 to 30 μM) in 50 mM Tris, pH 8.04 (RT), 150 mM NaCl, 0.5 mM EDTA, 0.001% Tween-20. Determination of the affinity constant was performed as described in [13 (link)].

Nicotinamide 1,N⁶-ethenoadenine dinucleotide (ε-NAD, Sigma-Aldrich) is a fluorescent analog of NAD⁺ and was utilized in kinetic assays as a TIR-1 substrate. TIR-1 cleaves the nicotinamide moiety from ε-NAD to release nicotinamide and etheno-ADPR (ε-ADPR), which fluoresces (λ_ex = 330 nm, λ_em = 405 nm). Enzymatic activity was assayed in Assay Buffer (50 mM Tris pH 8.0, 150 mM NaCl; final concentration) using Corning 96–well Half Area Black Flat Bottom Polystyrene NBS Microplates for a final reaction volume of 60 μL, or Corning 384-well Low Volume Black Round Bottom Polystyrene NBS Microplates for a final volume of 20 µL; reactions were initiated by the addition of ε-NAD. ε-ADPR fluorescence intensity readings were taken in real time every 15 s for 15–30 min using Wallac EnVision Manager Software and a PerkinElmer EnVision 2,104 Multilabel Reader. Fluorescence intensity readings (λ_ex = 330 nm, λ_em = 405 nm) were converted to [ε-ADPR] with an ε-ADPR standard curve, which was produced by incubating fixed concentrations (0–400 µM) of ε-NAD with excess ADP-ribosyl cyclase and plotting the peak fluorescence intensity values against [ε-ADPR]. The activity was linear with respect to time under all conditions tested.