Yes, samples may be stored for at least 1 month at -80 °C following neutralization - see step 5 of the kit protocol provided at the link below:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/234/464/mak460pis-ms.pdf
MAK460
NAD+/NADH Assay Kit
Sufficient for 100 fluorometric tests
Synonym(s):
NAD/NADH Quantitation Kit
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About This Item
NACRES:
NA.84
UNSPSC Code:
12161503
Pricing and availability is not currently available.
Quality Segment
application(s)
pharmaceutical
detection method
fluorometric
relevant disease(s)
cancer, Alzheimer′s disease; neurological disorders, Parkinson′s disease; aging/geriatric diseases
storage temp.
−20°C
General description
Pyridine nucleotides play an important role in metabolism and, thus, there is continual interest in monitoring their concentration levels. Quantitative determination of NAD+/NADH has applications in research pertaining to energy transformation and redox state of cells or tissue.
Pyridine nucleotides, including nicotinamide adenine dinucleotide (NAD), are crucial in cellular metabolism, impacting how energy is processed and managed within cells. Monitoring the concentrations of NAD and its reduced form, NADH, is vital for understanding cellular energy dynamics and redox balance. NAD, functioning as a central electron carrier, alternates between NAD+ (oxidized) and NADH (reduced) forms, facilitating numerous metabolic reactions. This transformation is essential not only for redox reactions but also for other metabolic functions, such as ADP-ribosylation, which is crucial for regulating protein functions and DNA repair. Additionally, NAD+ acts as a substrate for sirtuins, which are important for energy metabolism and modulating oxidative stress. .
Application
- Enhanced glycolysis in the myometrium with ectopic endometrium of patients with adenomyosis: a preliminary study.: This study investigates the role of glycolysis in the myometrium of patients with adenomyosis, utilizing the NAD+/NADH Assay Kit to assess metabolic alterations (Huang et al., 2024). 10.1080/09513590.2024.2332411
- Jian-Pi-Yi-Shen formula alleviates renal fibrosis by restoring NAD+ biosynthesis in vivo and in vitro.: This research demonstrates how the Jian-Pi-Yi-Shen formula mitigates renal fibrosis through NAD+ biosynthesis, with the NAD+/NADH Assay Kit playing a crucial role in the biochemical analysis (Gao et al., 2023). 10.18632/aging.205352
- Butyrate inhibits the mitochondrial complex Ι to mediate mitochondria-dependent apoptosis of cervical cancer cells.: The study explores butyrate′s effects on mitochondrial function and apoptosis in cervical cancer cells, employing the NAD+/NADH Assay Kit to measure mitochondrial activity (Zhang et al., 2023). 10.1186/s12906-023-04043-3
- PM2.5 mediates mouse testis Sertoli TM4 cell damage by reducing cellular NAD().: This article investigates the impact of PM2.5 on Sertoli cells in mouse testes, using the NAD+/NADH Assay Kit to track changes in cellular NAD levels (Xu et al., 2023). 10.1080/15376516.2023.2215862
- Synergistic Effect and Mechanism of Plumbagin with Gentamicin Against Carbapenem-Resistant Klebsiella pneumoniae.: The research highlights the synergistic antibacterial effects of plumbagin and gentamicin, with the NAD+/NADH Assay Kit being used to evaluate the metabolic impact on bacterial cells (Chen et al., 2020). 10.2147/IDR.S265753
The NAD+/NADH Assay Kit may be used for:
- Cancer Research
- Neurodegenerative Research
- Age/Geriatric Related Disease Research
The kit is suitable for the determination of NAD and NADH and the evaluation of drug effects on NAD/NADH metabolism in cells, tissue extracts etc.
Biochem/physiol Actions
The NAD+/NADH assay kit is based on a lactate dehydrogenase cycling reaction, in which the formed NADH reduces a probe into a highly fluorescent product. The fluorescence intensity of this product, measured at λEx = 530 nm/λEm = 585 nm, is proportional to the NAD+/NADH concentration in the sample. This assay is highly specific for NAD+/NADH with minimal interference (<1%) by NADP+/NADPH and is a convenient method to measure NAD, NADH and their ratio.
Features and Benefits
Broad Detection Range: Detect NAD and NADH levels accurately across a wide range, from 0.02 to 1 μM, using only 50 μL of sample in a 96-well plate setup, ensuring comprehensive analysis with minimal sample requirement
Simplified Process: Experience a streamlined process with the addition of only a single working reagent and a 10 minute room temperature reaction, reducing complexity and saving valuable time and effort.
Compatibility with High-Throughput Systems: Easily incorporate our kit into high-throughput handling systems, ensuring smooth and accurate processing, enhancing efficiency in your laboratory workflow.
Simplified Process: Experience a streamlined process with the addition of only a single working reagent and a 10 minute room temperature reaction, reducing complexity and saving valuable time and effort.
Compatibility with High-Throughput Systems: Easily incorporate our kit into high-throughput handling systems, ensuring smooth and accurate processing, enhancing efficiency in your laboratory workflow.
Other Notes
For additional information on our range of Biochemicals, please complete this form.
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This Item | |||
|---|---|---|---|
| Quality Level 100 | Quality Level - | Quality Level - | Quality Level - |
| detection method fluorometric | detection method colorimetric, fluorometric | detection method - | detection method - |
| storage temp. −20°C | storage temp. −20°C | storage temp. 2-8°C | storage temp. −20°C |
| application(s) pharmaceutical | application(s) pharmaceutical | application(s) - | application(s) - |
| relevant disease(s) cancer, Alzheimer′s disease; neurological disorders, Parkinson′s disease; aging/geriatric diseases | relevant disease(s) gastrointestinal diseases; cancer | relevant disease(s) - | relevant disease(s) - |
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Warning
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pcodes
Hazard Classifications
Met. Corr. 1
wgk
WGK 3
flash_point_f
Not applicable
flash_point_c
Not applicable
Storage Class
12 - Non Combustible Liquids
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At the end of sample preparation, can we store the supernatants in either -20 °C or -4 °C and use the supernatants later for this assay so that we can wait until we accumulate 96 samples for 96 wells?
1 answer-
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Can I use this kit on an already isolated mitochondria sample (using a different kit to isolate from brain tissue), or is this kit only viable on tissue that is homogenized in NAD+/NADH extraction buffer? Thank you!
1 answer-
This kit has not been validated for use with isolated mitochondria samples. However, the procedure for a discontinued NAD+/NADH assay kit suggests that mitochondria should be lysed in the Extraction Buffer included in this kit and then deproteinized using a 10 kDa spin column. Alternatively, 2% CHAPS in TBS (25 mM Tris, 0.15M NaCl; pH 7.4) may be used to lyse the purified mitochondria. Vortexing the pellet for 1 minute followed by centrifugation at high speed for 2 minutes will yield a supernatant containing soluble mitochondrial protein. A similar procedure may be applicable for testing isolated mitochondria samples with this product.
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How can I determine the shelf life / expiration / retest date of this product?
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If this product has an expiration or retest date, it will be shown on the Certificate of Analysis (COA, CofA). If there is no retest or expiration date listed on the product's COA, we do not have suitable stability data to determine a shelf life. For these products, the only date on the COA will be the release date; a retest, expiration, or use-by-date will not be displayed.
For all products, we recommend handling per defined conditions as printed in our product literature and website product descriptions. We recommend that products should be routinely inspected by customers to ensure they perform as expected.
For products without retest or expiration dates, our standard warranty of 1 year from the date of shipment is applicable.
For more information, please refer to the Product Dating Information document: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/418/501/product-dating-information-06-25-mk.pdfHelpful?
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This kit can be used for detection of NAD+/NADH levels in yeast culture?
1 answer-
This kit has not been specifically tested for use in detecting NAD and NADH in yeast extract or culture broth, but it likely could be used for either one. While a specific method for yeast is not provided, please see the Sample Preparation section on page 2 of the Technical Bulletin for this kit, for general recommendations.
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How is shipping temperature determined? And how is it related to the product storage temperature?
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Products may be shipped at a different temperature than the recommended long-term storage temperature. If the product quality is sensitive to short-term exposure to conditions other than the recommended long-term storage, it will be shipped on wet or dry-ice. If the product quality is NOT affected by short-term exposure to conditions other than the recommended long-term storage, it will be shipped at ambient temperature. As shipping routes are configured for minimum transit times, shipping at ambient temperature helps control shipping costs for our customers. For more information, please refer to the Storage and Transport Conditions document: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/316/622/storage-transport-conditions-mk.pdf
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Can this kit be used for detection of NADH levels in bacteria culture?
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This kit has not been tested for use on bacterial cultures and may not be suitable. It would be up to the end user to determine suitability. However, the colorimetric version of this kit, MAK468, has been successfully used in Corynebacterium glutamicum, Klebsiella pneumoniae, Lactobacillus plantarum, Edwardsiella tarda, Acinetobacter baumannii, Auxenochlorella protothecoides, E. coli, and other microbial species. Please see the links below to review this product and protocol.
MAK468 product page:
https://www.sigmaaldrich.com/product/sigma/mak468
MAK468 datasheet:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/219/480/mak468pis-ms.pdfHelpful?
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Does this assay measure total NAD+/NADH concentrations? If so, How can the conversion to NAD+ or NADH only be achieved?
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The calculation method is the same for NAD+ and NADH. However, there should be one well for the NAD+ test and another well for the NADH test. If the intention is to solely measure NAD+, the sample should be processed according to the NAD+ instructions. This involves homogenizing the sample with NAD+ extraction buffer, then proceeding with heating and addition of the assay buffer followed by the addition of the opposite buffer (in this case, NAD extraction buffer). The order of addition of the extraction buffers determines whether NAD+ or NADH is being measured, as it degrades the other due to the extraction pH.
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Can a white plate be used instead of a black plate for this assay?
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A black, opaque, flat-bottomed plate is recommended for fluorescence assays, and a black plate with a clear bottom is also acceptable. White plates are not recommended for fluorescence assays as they can increase background and cross-talk. An example of a recommended plate type for fluorescence assays is the product M9685.
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Is this kit's sample only for cultured cells? Do you have any data or protocols for serum, urine, saliva, etc.?
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This kit has not been tested with serum or urine samples. Unfortuantely, publications regarding this application could not found.
However, there may not be any problems with using plasma, as well as serum, with the kit. It is prudent to test a number of dilutions of urine to determine the best concentration to use in the assay. The following substances interfere with the assay and should be avoided in sample preparation: EDTA (>0.5 mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).Helpful?
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