Cell lysate from a cell culture can be easily prepared via Deproteinzation using the extraction buffer and filtration tubes found within this kit. Intracellular NADH levels can be quantified by heat treatment of cell lysate. Additionally, intracellular NAD+ levels can be determined by subtracting NADH levels from independently measured total NAD+/NADH levels.
NAD/NADH Assay Kit-WST enables quantification of the amount of total NAD+/NADH, NADH and NAD+ in cells and measurement of their ratio.
Measurement of NAD+ and NADH
Cell lysate from a cell culture can be easily prepared via Deproteinzation
using the extraction buffer and
filtration tubes found within this kit.
Intracellular NADH levels can be quantified by heat treatment of cell lysate. Additionally, intracellular NAD+ levels can be
determined by subtracting
NADH levels from independently measured total NAD+/NADH levels.
・Study of NAD+/NADH as Markers
Recently, it has become clear that Sirtuin is linked to longevity and plays a role in NAD+ level regulation. Also Sirtuin has been recognized as a marker necessary to understanding biological states, such as obesity & diabetes, as well as cellular differentiation.
In the experiment shown below, the NAD+ /NADH levels & ratio were determined using HeLa cells.
Standard curves were constructed using different concentrations of HeLa cells (2.5×105 and 5.0×105 cells) cultured in growth media. The standard curves were then used to determine the intracellular NAD+ and NADH levels. As a result, NAD+ and NADH levels varied depending on cell number while the change in cell number had no effect on the NAD+/NADH ratio.
Measurement of NAD+/NADH in Combination with Lactate Assay Kit
Change in metabolic activity was observed when the glycolytic inhibitor 2-Deoxy-D-glucose was added to HeLa cells.
2-Deoxy-D-glucose was added to HeLa cells (1×106 cells) to obtain a final concentration of 6 mmol/l 2-Deoxy-D-glucose. After 24 hours of incubation, lactate levels in the supernatant were quantified using the Lactate Assay Kit-WST (Item#: L256), and the NAD+/NADH ratio was determined with the cell pellet after removing the supernatant using the NAD/NADH Assay Kit-WST.
As a result, intracellular glycolysis was inhibited by 2-Deoxy-D-glucose, which led to decreased lactate levels and an increase in the NAD+/NADH ratio.
Q. How many samples can I measure?
A. The number of samples that can be measured when the standard sample is serially diluted from 2 μmol / l is shown in the table above.* The number of samples that can be recorded when the sample is measured in triplicates.
It is necessary to create a calibration curve per measurement when you perform the measurement separately. If the measurement is done separately, the above sample number will be less.
Q. I do not have a 450 nm filter. What other filters can I use?
A. You can use filters with an absorbance between 450 nm and 490 nm. However, the absorbance value can become lower if the sample is not measured at 450 nm.
Q. Can I purchase the filtration tube separately?
A. No, we don’t sell the filtration tube included in the kit separately. If you need additional supplies, you can also use a commercially available filtration tube.
Product：Nanosep® MF Centrifugal Devices (MWCO：10K, color：blue)
Q. How stable is Working solution?
Ａ. You can’t store the Working solution. Please prepare the Working solution prior to use. Please protect from light because the Working solution ins light-sensitive. The Working solution is stable for 4 hours at room temperature with protection from light.
Q. Our samples did not change in color, are there any reasons for this?
A. NAD level contained in the sample may be lower than the detection limit that can be measured using this kit.
Please increase the number of cells or lower the dilution ratio if you dilute the sample.