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MAK037

Sigma-Aldrich

NAD/NADH Quantitation Kit

NAD/NADH Quantitation Kit

sufficient for 100 colorimetric tests

Synonym(s):

NAD/NADH Assay Kit

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About This Item

UNSPSC Code:
12161503
eCl@ss:
32161000
NACRES:
NA.84

usage

sufficient for 100 colorimetric tests

detection method

colorimetric

relevant disease(s)

cancer

storage temp.

−20°C

General description

Nicotinamide adenine dinucleotide (NAD) is an enzymatic cofactor involved in many redox reactions. NAD functions as an electron carrier, cycling between the oxidized (NAD) and reduced (NADH) forms.[1] In addition to its role in redox reactions, NAD plays critical roles in ADP (adenosine diphosphate)-ribosylation reactions and as a substrate for sirtuins.[2]

Application

NAD/NADH Quantitation Kit has been used to quantify NAD+/NADH influenced by SIRT1 (sirtuin 1) activity (associated with ischemia-reperfusion injury) in the presence of trimetazidine.[3]

Suitability

Suitable for the detection of NADH and NAD in animal tissues (liver, kidney etc.), cell culture (adherent or suspension cells), serum or urine.

Principle

The NAD/NADH Quantification Kit provides a convenient tool for sensitive detection of the intracellular nucleotides, NAD (nicotinamide adenine dinucleotide) and NADH, and their ratio without the requirement to purify NAD/NADH from samples. This assay is specific for NAD and NADH, and does not detect NADP or NADPH. NADt (NAD and NADH) or NADH are quantified in a colorimetric assay (450 nm).

Related product

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Description
Pricing

Pictograms

Exclamation mark
GHS07

signalword

Warning

hcodes

H315,H319

Hazard Classifications

Eye Irrit. 2 - Skin Irrit. 2

Storage Class

10 - Combustible liquids

Choose from one of the most recent versions:

Certificates of Analysis (COA)

Lot/Batch Number
8J09K03370
8A17K03370
8B09K03370
8G29K03370
8H24K03370

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Frédérique Moyrand et al.
Eukaryotic cell, 7(12), 2069-2077 (2008-09-30)
The genome of the basidiomycete pathogenic yeast Cryptococcus neoformans carries two UDP-glucose epimerase genes (UGE1 and UGE2). UGE2 maps within a galactose cluster composed of a galactokinase homologue gene and a galactose-1-phosphate uridylyltransferase. This clustered organization of the GAL genes
Deepanwita Banerjee et al.
BMC systems biology, 11(1), 51-51 (2017-04-28)
The leading edge of the global problem of antibiotic resistance necessitates novel therapeutic strategies. This study develops a novel systems biology driven approach for killing antibiotic resistant pathogens using benign metabolites. Controlled laboratory evolutions established chloramphenicol and streptomycin resistant pathogens
Andrea M Lencina et al.
mBio, 9(4) (2018-07-05)
The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn's first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme
Haiping Wang et al.
Nature immunology, 21(3), 298-308 (2020-02-19)
Depleting regulatory T cells (Treg cells) to counteract immunosuppressive features of the tumor microenvironment (TME) is an attractive strategy for cancer treatment; however, autoimmunity due to systemic impairment of their suppressive function limits its therapeutic potential. Elucidating approaches that specifically
Mammalian sirtuins?emerging roles in physiology, aging, and calorie restriction.
Haigis M C and Leonard P G
Genes & Development, 20(21), 2913-2921 (2006)
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1 Review This action will navigate to reviews.

Questions

1–9 of 9 Questions  

  1. How is shipping temperature determined? And how is it related to the product storage temperature?

    1 answer

    1. 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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  2. How can I determine the shelf life / expiration / retest date of this product?

    1 answer

    1. 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/449/386/product-dating-information-mk.pdf

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  3. Can this kit be used to quantify NADH, NADHt levels in mammalian mitochondria?

    1 answer

    1. This kit may be used with purified mitochondria and sub-mitochondrial particles provided the concentration is above 200nM. It is recommended that the mitochondria 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 mitochondrial. Vortex the pellet for 1 minute then centrifuge mitochondria at high speed for 2 minutes. The supernatant contains soluble mitochondrial protein. Please see the link below to review the product datasheet:
      https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/299/569/mak037bul.pdf

      Please see the link below to review an Amicon Ultra 10 kDa centrifugal filter:
      https://www.sigmaaldrich.com/product/mm/ufc5010

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  4. Is the molecular weight of NADH required for the ratio calculation? Also, is it necessary to conduct both assays for total NAD/NADH and NADH only?

    1 answer

    1. The molecular weight is not necessary for calculating the ratio, as only the pmole from the standard curve is used in the calculation. However, the NADH molecular weight is provided in case there is a need to convert from pmole to grams.
      The kit offers the option to measure NAD total (NAD+ and NADH) or NADH only. If the goal is to determine the ratio, both the total NAD+NADH assay and the NADH-only detection should be conducted. However, if the aim is to determine the concentration of total (NAD + NADH) or NADH only without the ratio, there is no need to perform both assays.
      The NAD content can be determined by subtracting NAD total from NADH content, and the value from the standard curve will be in pmole.

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  5. Are the extraction buffers in MAK037A and MAK038A identical? Can the NADH extraction buffer in Product No. MAK037 be used for other assays besides quantifying enzyme activity?

    1 answer

    1. The composition of the extraction buffers for MAK037 and MAK038 is proprietary. However, they are reported to contain bicarbonate and detergent with a pH of approximately 10, making them technically interchangeable. The specific composition of the Extraction Buffer is confidential and is intended to preserve the redox state of the lysate for assaying the NAD/NADH ratio. While the buffer is suitable for BCA assays for protein quantification, its compatibility with enzymatic assays is not confirmed. It is not recommended for standard enzyme activity assays due to potential issues with specific enzymes. Therefore, if considering its use for this purpose, it is suggested to compare its performance with that of a standard lysis buffer for enzyme activity.

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  6. Is the NAD kit capable of measuring NAD levels in whole blood?

    1 answer

    1. The MAK037 kit has not been specifically tested or used to measure NAD levels in whole blood samples. However, the scientific literature reveals a reference indicating that researchers have used this kit with a whole blood sample (Wakade C, Chong R, Bradley E, Thomas B, Morgan J, 2014, "Upregulation of GPR109A in Parkinson’s Disease," PLOS ONE 9(10): e109818). It is anticipated that the kit may be suitable for such a sample, but this has not been directly verified.

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  7. Is the components of the extraction buffer proprietary or can I know what it is composed of

    1 answer

    1. The components of the extraction buffer in MAK037 are proprietary.

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  8. Question for the product MAK037-1KT, NAD/NADH Quantitation Kit: Is it possible to use the kit for examination of bacterial cultures samples? 

    1 answer

    1. This kit has not been specifically tested for use with bacterial cultures. It has been qualified for use in mammalian cells, tissues, serum, or urine. Provided that bacteria are thoroughly lysed and NAD/NADH levels are sufficient, there is no reason to suspect that this kit would not be suitable. Please see the link below to review product datasheet:
      https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/299/569/mak037bul.pdf

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  9. What is the lowest limit of detection for this kit? I am trying to use very small amount of tissue from mouse embryo (~1mg) and would like to know if that would be possible. Thanks!

    1 answer

    1. The detection range would be roughly 0.4 µM (0.4 pmol/µl) - 20 µM (20 pmol/µl).
      The lower limit of detection is calculated by taking 20 pmole (lowest concentration of Standard) divided by 50 µl (max sample volume) =20 pmol/50µl = 0.4 pmol/µl = 0.4 µM.

      Please see the product information sheet to know more about the details of this product:
      https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/299/569/mak037bul.pdf

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Reviews

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    • Review 1
    • Votes 0
    5 out of 5 stars.

    NAD/NADH Quantitation Kit

    It works well in the mouse kidney tissue. The lysates need to diluent for the kit, otherwise, it will overrange.

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