Amplite® Fluorimetric cADP-Ribose Assay Kit

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.

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.

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.

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.

Table 2. Reagent composition for each well.

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.