NAD/NADH Ratio Assay Kit (Red Fluorescence)
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Catalog Number:
CA-N226
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Detailed Description
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CA-N226
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Name
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Elite™ NAD/NADH Ratio Assay Kit (Red Fluorescence)
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Description
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This Fluorimetric NAD/NADH Ratio Assay Kit provides a convenient method for sensitive detection of NAD, NADH and their ratio; there is no need to purify NAD/NADH from sample mix. It has very low background since it is run in the red visible range that significantly reduces the interference resulted from biological samples.
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Application
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The assay can be conveniently performed in a 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 530 - 570/590 - 600 nm (maximum Ex/Em = 540/590 nm) or an absorbance microplate reader at ~576 nm. This kit provides NAD and NADH extraction buffer, and cell lysis buffer for your convenience. It has been frequently used for determining NAD/NADH from cell lysates.
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Size
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250 assays in 96-well plates
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Ex/Em
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540/590 nm
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Detection
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Fluorescence microplate reader
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For Downloading
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===> DATA SHEET ===> MSDS
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Selected Citations:
1) Suppressed expression of LDHB promotes age-related hearing loss via aerobic glycolysis. Cell Death Dis. 11, 1–13 (2020).
2) Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells. Toxicol. Sci. 153, 341–351 (2016).
Frequently Asked Questions
1. Can we use this ratio assay kit to also detect NAD or NADH separately?
Answer: Yes. You could measure the NADH amount with Component D (NADH Extraction Solution), and measure the NAD amount with NAD Extraction Solution. But according to our experience, the NAD concentrations in cells are a lot higher than the NADH concentrations. We suggest to measure the Total NAD+NADH amount and the NAD amount, then to calculate the NADH amount by subtraction, which gives more accurate results.
2. Why PBS is used to dilute the Standard? Since the samples are in the lysis buffer, do you think I should use the lysis buffer to dilute them?
Answer: PBS is commonly used. People usually use the lysis buffer to lyse the cells, then use PBS to dilute the samples. The point is that you should keep the same concentration of the lysis buffer for the samples and the Standards.
3. I did a comparison, and it showed that the PBS based Standard curve looked better than the lysis buffer based Standard curve. What do you think of this?
Answer: The bubbles in the lysis buffer may cause the inconsistency of the readings.
4. When I do duplicates of Standards, the reading varies a lot in the low range (0.2 uM or lower). Why does that happen?
Answer: It is normal. There are many factors that would cause inconsistent readings, i.e. bubbles in the wells, the volumes.... So try doing more replicates.
5. Sometimes for the 2 sets of Standards I set up, they look good (R2=0.99) individually but the curve is messed up (R2=0.95) when merged. So which curve should I use to quantify my data?
Answer: You'll need to use the curve set up on the same plate with your samples.
6. We don't have fluorescence microplate reader, so can we use absorbance microplate reader?
Answer: Yes. But the absorbance readings have much lower sensitivity compared to the fluorescence readings. Try using the ratio of 570 to 610 to increase the sensitivity.
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