CD38 activity was measured using the nicotinamide 1,N6-ethenoadenine dinucleotide (ε-NAD) fluorescence-based enzymatic assay, as described previously (83 ). Primary astrocyte cells were incubated with ε-NAD (40 μM; Sigma, N5131) in an extracellular solution of Hanks′ Balanced Salt solution (HBSS) with Ca+2/Mg+2. Following a 15-min incubation, fluorescence was read at λEx = 300 nm and λEm = 410 nm using a fluorescence microplate reader (Synergy HT, BioTek). The fluorescent readings were then normalized to viable cell numbers as determined by the AlamarBlue assay (Bio-Rad, #BUF012B). Normalized values are presented as CD38 activity.
ε nad
Manufactured by Merck Group
ε-NAD is a laboratory product manufactured by Merck Group. It is a cofactor that serves as a key component in various enzymatic reactions. The core function of ε-NAD is to facilitate the transfer of electrons during oxidation-reduction processes in biological systems.
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Lab products found in correlation
Synergy h1 hybrid multi mode reader, by Agilent Technologies (2 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Alamar blue assay, by Bio-Rad (1 mentions)
96 well half area black flat bottom polystyrene nbs microplates, by Corning (1 mentions)
384 well low volume black round bottom polystyrene nbs microplates, by Corning (1 mentions)
Envision manager software, by PerkinElmer (1 mentions)
Envision 2104 multilabel reader, by PerkinElmer (1 mentions)
N2630, by Merck Group (1 mentions)
Infinite m200 plate reader, by Tecan (1 mentions)
Prism 9, by GraphPad (1 mentions)
Ni nta agarose resin, by Thermo Fisher Scientific (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
7 protocols using ε nad
1
Fluorescence-Based CD38 Activity Assay
2
Fluorometric TIR-1 Enzymatic Assay
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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 (λex = 330 nm, λem = 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 (λex = 330 nm, λem = 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.
3
Enzymatic NADase Activity Assay
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The NADase reaction was performed in black 96-well half area plates (Corning, 3694). In each microwell, ThsA protein which was purified as previously described (Ofir et al., 2021), was added to cell lysate or to a positive control of 100 mM sodium phosphate buffer pH 8 supplemented with 1′′–3′ gcADPR standards. ThsA was added to a final concentration of 100 nM protein in a 50 μl final volume reaction. Five microlitres of 5 mM nicotinamide 1,N6 -ethenoadenine dinucleotide (εNAD, Sigma,N2630) solution was added to each well immediately before the beginning of measurements and mixed by pipetting to reach a concentration of 500 μM in the 50 μl final reaction volume. Plates were incubated inside a Tecan Infinite M200 plate reader at 25°C, and measurements were taken every 1 min at 300 nm excitation wavelength and 410 nm emission wavelength. Reaction rate was calculated from the linear part of the initial reaction.
4
Fluorometric assay for NADase activity
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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.
5
In vitro Rtn4 ubiquitination assay
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In vitro Rtn4 ubiquitination assays were described previously.15 (link) For linking recombinant SdeC derivatives to agarose beads, Ni–NTA agarose resin (Thermo Fisher) was washed 3× with 1× ART buffer (20 mM Tris, 10 mM NaCl, pH 7.4) and mixed with His6-SdeC followed by incubation for 1 h at 4 °C. Next, the SdeC-bound agarose resin was washed 3× with 1× ART buffer before processing to the two-step bead assay. During preincubation, SdeC-bound agarose beads were added to 10 mM Ub, and 100 mM nicotinamide 1, N6-ethenoadenine dinucleotide (ε-NAD, Sigma) in 1× ART buffer (SdeC final concentration 50 nM), and incubated for 1 h at 37 °C. After incubation, the beads were removed from reaction by spin filter columns. The supernatant containing modified Ub and excess ε-NAD were mixed with 400 nM GST-HA-Rtn4 and fresh unbound SdeC variants, then incubated for another hour at 37 °C. Reactions were terminated by addition of reducing loading buffer and boiling.
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In vitro Rtn4 ubiquitination assays were described previously.15 (link) For linking recombinant SdeC derivatives to agarose beads, Ni–NTA agarose resin (Thermo Fisher) was washed 3× with 1× ART buffer (20 mM Tris, 10 mM NaCl, pH 7.4) and mixed with His6-SdeC followed by incubation for 1 h at 4 °C. Next, the SdeC-bound agarose resin was washed 3× with 1× ART buffer before processing to the two-step bead assay. During preincubation, SdeC-bound agarose beads were added to 10 mM Ub, and 100 mM nicotinamide 1, N6-ethenoadenine dinucleotide (ε-NAD, Sigma) in 1× ART buffer (SdeC final concentration 50 nM), and incubated for 1 h at 37 °C. After incubation, the beads were removed from reaction by spin filter columns. The supernatant containing modified Ub and excess ε-NAD were mixed with 400 nM GST-HA-Rtn4 and fresh unbound SdeC variants, then incubated for another hour at 37 °C. Reactions were terminated by addition of reducing loading buffer and boiling.
6
Fluorometric Assay for NADase Activity
7
NAD Cleavage Assay in Worms
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Cleavage of NAD is conveniently measured using 1,N6-etheno-NAD (ε-NAD, Sigma, N2630) since hydrolysis of this molecule can be detected fluorometrically (with excitation at 300 nm and emission at 410 nm) [26 (link),30 (link)]. Live adult worms (individually or in groups of two) were washed 3x with assay buffer (50 mM Tris-HCl [pH 9.0], 120 mM NaCl, 5 mM KCl, 50 mM glucose and 2 mM CaCl2) then incubated in assay buffer containing 500 µM ε-NAD in a final volume of 200 µl at 37°C. Changes in fluorescence were followed over time using a Synergy HT microplate reader (BioTek). The minimal background (substrate-only control) values have been subtracted from all data points which are reported as relative fluorescence units (RFU) per individual worm.
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