Amplite® Fluorimetric cADP-Ribose Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Platform
Fluorescence microplate reader
Excitation | 420 nm |
Emission | 480 nm |
Cutoff | 435 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare and add cADPR standards and/or test samples (50 µL)
- Prepare and add ADRPC working solution (50 µL)
- Incubate at room temperature for 1 hour
- Add 40 µL Quest Fluor™ NAD Probe
- Add 40 µL Assay Solution
- Incubate at room temperature for 20 minutes
- Add 30 µL Enhancer Solution
- Incubate at room temperature for 20 minutes
- 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
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
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.
BL | BL | TS | TS |
ST1 | ST1 | ... | ... |
ST2 | ST2 | ... | ... |
ST3 | ST3 | ||
ST4 | ST4 | ||
ST5 | ST5 | ||
ST6 | ST6 | ||
ST7 | ST7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
ST1-ST7 | 50 µL | Serial Dilution |
BL | 50 µL | Assay Solution I (Component C) |
TS | 50 µL | Test Sample |
NAD generation assay
- 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).
- 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.
- Incubate the reaction at room temperature for 60 minutes, protected from light.
NAD detection assay
- 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.
- 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.
- Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.
- 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.
- Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.
Images
Citations
Authors: Caldwell, Blake A and Wu, Yajun and Wang, Jing and Li, Liwu
Journal: Cell Reports (2024)
Authors: Sarkar, Ankita
Journal: (2024)
Authors: Sarkar, Ankita and Dutta, Sourav and Sur, Malinki and Chakraborty, Semanti and Dey, Puja and Mukherjee, Piyali
Journal: The FEBS Journal (2022)
References
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
Authors: Kuz'min, VS and Sosulina, LIu and Sukhova, GS and Ashmarin, IP
Journal: Uspekhi fiziologicheskikh nauk (2006): 3--17
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
Authors: Cheung, Donald W
Journal: European journal of pharmacology (2003): 57--59
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
Authors: Graeff, Richard and Lee, Hon Cheung
Journal: Biochemical Journal (2002): 379--384
Authors: Lukyanenko, Valeriy and Györke, Inna and Wiesner, Theodore F and Györke, S and or, undefined
Journal: Circulation research (2001): 614--622
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
Authors: THOMAS, Justyn M and MASGRAU, Roser and CHURCHILL, Grant C and GALIONE, Antony
Journal: Biochemical Journal (2001): 451--457
Authors: Chini, Eduardo N
Journal: Journal of Applied Physiology (2001): 516--521
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FAQ
How should I reconstitute an NADPH standard?
Will Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence* work with NADP/NADPH? Can this kit measure NADP+ and NADPH?
What is the concentration of calcium inside cells?
What assay kits measure NADP/NADPH from cell samples?