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Amplite® Fluorimetric cADP-Ribose Assay Kit

The concentration of cADPR was detected using Amplite® Fluorimetric cADP-Ribose Assay Kit. Different concentrations of cADPR were incubated with ADPRC reaction mix for 20 min at RT before NAD detection reagent was added. The lowest detected concentration of cADPR is 100 nM.
The concentration of cADPR was detected using Amplite® Fluorimetric cADP-Ribose Assay Kit. Different concentrations of cADPR were incubated with ADPRC reaction mix for 20 min at RT before NAD detection reagent was added. The lowest detected concentration of cADPR is 100 nM.
The concentration of cADPR was detected using Amplite® Fluorimetric cADP-Ribose Assay Kit. Different concentrations of cADPR were incubated with ADPRC reaction mix for 20 min at RT before NAD detection reagent was added. The lowest detected concentration of cADPR is 100 nM.
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


cADP-ribose (cADPR) is a novel Ca2+ messenger derived from NAD+. ADP-riboxyl cyclase (ADPRC) catalyzes the synthesis of cADPR from NAD+, but the reaction can be reversed in the presence of high concentration of nicotinamide, producing NAD+ from cADPR stoichiometrically. The resultant NAD+ can be detected using our newly developed NAD sensor Quest Fluor™ NAD reagent. This makes monitoring cADPR in tissues and cell cultures possible in the low nM range. The NAD+ detection using Quest Fluor™ NAD reagent is specific to NAD+ and has no reaction to NADH. The fluorescent signal can be readily detected. This assay can be performed in a convenient 96-well or 384-well microtiter plate.

Platform


Fluorescence microplate reader

Excitation420 nm
Emission480 nm
Cutoff435 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare and add cADPR standards and/or test samples (50 µL)
  2. Prepare and add ADRPC working solution (50 µL)
  3. Incubate at room temperature for 1 hour
  4. Add 40 µL Quest Fluor™ NAD Probe
  5. Add 40 µL Assay Solution
  6. Incubate at room temperature for 20 minutes
  7. Add 30 µL Enhancer Solution
  8. Incubate at room temperature for 20 minutes
  9. Monitor fluoroscence intensity at Ex/Em = 420/480 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

cADPR standard stock solution (5 mM):
Add 10 µL of ddH2O into the vial of cADPR standard (Component F) and mix them well. Note: The unused cADPR stock solution should be stored at -20°C in single use aliquots.

PREPARATION OF STANDARD SOLUTION

cADPR standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/20305

Note: Prepare cADPR serial dilutions in Assay Solution I (Component C).

PREPARATION OF WORKING SOLUTION

ADPRC working solution:
Add 50 µL ddH2O into the vial of ADPRC Enzyme Mix (Component B) and mix well. Transfer whole content into 5 mL of Assay Solution I (Component C) and mix them well. Note: ADPRC working solution is not stable, use it promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of cADPR standards and test samples in a solid black 96-well microplate. ST = cADPR standard (ST1-ST7); BL = blank control; TS = test sample.

BLBLTSTS
ST1ST1......
ST2ST2......
ST3ST3  
ST4ST4  
ST5ST5  
ST6ST6  
ST7ST7  

Table 2. Reagent composition for each well.

WellVolumeReagent
ST1-ST750 µLSerial Dilution
BL50 µLAssay Solution I (Component C)
TS50 µLTest Sample

NAD generation assay

  1. Add 50 µL of cADPR standard, blank control, and test samples to solid black 96-well microplate (As shown in Table 1 and Table 2).

  2. Add 50 µL/well of ADPRC working solution into each well of cADPR standard, blank control and test samples. Note: For a 384-well plate, add 12.5 µL of sample and 12.5 µL of ADPRC Reaction Mix Solution into each well.

  3. Incubate the reaction at room temperature for 60 minutes, protected from light.

NAD detection assay

  1. Add 40 µL Quest Fluor™ NAD Probe (Component A) into each well of cADPR standard, blank control, and test samples (total of 140 µL/well), mix well.

  2. Add 40 µL Assay Solution II (Component D) into each well (total of 180 µL/well), mix well. Note: For a 384-well plate, add 10 µL of Quest Fluor™ NAD Probe and 10 µL Assay Solution II into each well.

  3. Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.

  4. Add 30 µL Enhancer Solution (Component E) to each well to make the total NAD assay volume of 210 µL/well, and incubate at room temperature for 10-20 minutes, protected from light. Note: For a 384-well plate, add 7.5 µL Enhancer Solution.

  5. Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.

Images


Citations


View all 3 citations: Citation Explorer
Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis
Authors: Caldwell, Blake A and Wu, Yajun and Wang, Jing and Li, Liwu
Journal: Cell Reports (2024)
Early loss of endogenous NAD+ following rotenone treatment leads to mitochondrial dysfunction and Sarm1 induction that is ameliorated by PARP inhibition
Authors: Sarkar, Ankita and Dutta, Sourav and Sur, Malinki and Chakraborty, Semanti and Dey, Puja and Mukherjee, Piyali
Journal: The FEBS Journal (2022)

References


View all 35 references: Citation Explorer
Ca 2+ release induced by cADP-ribose is mediated by FKBP12. 6 proteins in mouse bladder smooth muscle
Authors: Zheng, Ji and Wenzhi, Bi and Miao, Lin and Hao, Yumin and Zhang, Xu and Yin, Wenxuan and Pan, Jinhong and Yuan, Zengqiang and Song, Bo and Ji, Guangju
Journal: Cell calcium (2010): 449--457
ADP-ribose and cADP-ribose--endogenous regulators of cellular ionic balance. Cardiotropic effects of ADP-ribose
Authors: Kuz'min, VS and Sosulina, LIu and Sukhova, GS and Ashmarin, IP
Journal: Uspekhi fiziologicheskikh nauk (2006): 3--17
Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes
Authors: Fritz, Nicolas and Macrez, Nathalie and Mironneau, Jean and Jeyakumar, Loice H and Fleischer, Sidney and Morel, Jean-Luc
Journal: Journal of cell science (2005): 2261--2270
Modulation of spontaneous transient Ca 2+-activated K+ channel currents by cADP-ribose in vascular smooth muscle cells
Authors: Cheung, Donald W
Journal: European journal of pharmacology (2003): 57--59
Effect of halothane on cADP-ribose-induced Ca2+ release system in tracheal smooth muscle
Authors: Chini, Eduardo N and Keller, Thomas F and Prakash, Yedatore S and Pabelick, Christina M and Sieck, Gary
Journal: Anesthesiology: The Journal of the American Society of Anesthesiologists (2002): 1022--1024
A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity
Authors: Graeff, Richard and Lee, Hon Cheung
Journal: Biochemical Journal (2002): 379--384
Potentiation of Ca2+ release by cADP-ribose in the heart is mediated by enhanced SR Ca2+ uptake into the sarcoplasmic reticulum
Authors: Lukyanenko, Valeriy and Györke, Inna and Wiesner, Theodore F and Györke, S and or, undefined
Journal: Circulation research (2001): 614--622
Functional overlap of IP3-and cADP-ribose-sensitive calcium stores in guinea pig myenteric neurons
Authors: Turner, Douglas J and Segura, Bradley J and Cowles, Robert A and Zhang, Weizhen and Mulholl, undefined and , Michael W
Journal: American Journal of Physiology-Gastrointestinal and Liver Physiology (2001): G208--G215
Pharmacological characterization of the putative cADP-ribose receptor
Authors: THOMAS, Justyn M and MASGRAU, Roser and CHURCHILL, Grant C and GALIONE, Antony
Journal: Biochemical Journal (2001): 451--457
Selected contribution: effect of volatile anesthetics on cADP-ribose-induced Ca2+ release system
Authors: Chini, Eduardo N
Journal: Journal of Applied Physiology (2001): 516--521