概述

  • 产品名称
    NAD/NADH Assay试剂盒(Colorimetric)
    参阅全部 NAD/NADH 试剂盒
  • 检测方法
    Colorimetric
  • 样品类型
    Tissue Extracts, Cell Lysate
  • 检测类型
    Quantitative
  • 检测时间
    2h 00m
  • 种属反应性
    与反应: Mammal
  • 产品概述

    NADH/NAD Quantification Kit (Colorimetric) (ab65348) provides a convenient tool for sensitive detection of the intracellular nucleotides: NADH, NAD and their ratio. Assay of nicotinamide nucleotides is of continual interest in the studies of energy transforming and redox state of cells or tissues.


    The NAD Cycling Enzyme Mix in the kit specifically recognizes NADH/NAD in an enzyme cycling reaction. There is no requirement to purify NADH/NAD from samples. The reaction specifically detects NAD and NADH, but not NADP nor NADPH. The enzyme cycling reaction significantly increases the detection sensitivity and specificity. NADt (NAD and NADH) or NADH can be easily quantified by comparing with standard NADH.


    Visit our FAQs page for tips and troubleshooting.


    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • 说明

    If you would like to use a fluorometric reading, please refer to NAD/NADH Assay Kit (Fluorometric) (ab176723).

  • 经测试应用
    适用于: Functional Studiesmore details
  • 平台
    Microplate

性能

  • 存放说明
    Store at -20°C. Please refer to protocols.
  • 组件 标识符 100 tests
    NAD Cycling Buffer NM 1 x 15ml
    NAD Cycling Enzyme Mix Green 1 vial
    NADH Developer Purple 1 vial
    NADH Standard (MW:663.4) Yellow 1 x 200nmole
    NADH/NAD Extraction Buffer NM 1 x 50ml
    Stop Solution Red 1 x 1.2ml
  • 研究领域
  • 相关性
    NAD (Nicotinamide adenine dinucleotide) is a coenzyme in metabolic redox reactions, a precursor for several cell signaling molecules, and a substrate for protein posttranslational modifications. NAD is a dinucleotide, consisting of two nucleotides joined through their phosphate groups: with one nucleotide containing an adenosine ring, and the other containing nicotinamide. In metabolism, NAD is involved in redox reactions, carrying electrons from one reaction to another. The coenzyme is therefore found in two forms in cells: NAD is an oxidizing agent – it accepts electrons from other molecules and becomes reduced, forming NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD. However, it is also used in other cellular processes, the most notable one being a substrate of enzymes that add or remove chemical groups from proteins in posttranslational modifications.

相关产品

应用

Our Abpromise guarantee covers the use of ab65348 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
Functional Studies Use at an assay dependent dilution.

图片

  • Functional Studies - NAD/NADH Assay Kit (ab65348)
    Functional Studies - NAD/NADH Assay Kit (ab65348)
    NAD/NADH was measured in K562 ME2 knockdown cells(pLKO - empty vector; shME2-2 & shME2-3 - two selected knockdown clones). Data are expressed as mean ±: SD, n=3. NAD/NADH Ratio is calculated as described in the product protocol.

    Image obtained from Ren JG et al; PLOS one, 2010; 5(9): e12520 (DOI:10.1371/journal.pone.0012520)

  • Functional assay: NAD/NADH Assay Kit (ab65348)
    Functional assay: NAD/NADH Assay Kit (ab65348)

    NAD and NADH (tNAD) or NADH alone measured cell lysates. 5e6 cells were lysed in 1 mL, spin filtered, and tested neat or 1/5 (duplicates +/- SD).

  • Functional assay: NAD/NADH Assay Kit (ab65348)
    Functional assay: NAD/NADH Assay Kit (ab65348)

    Standard curve with background signal subtracted (duplicates; +/- SD).

  • Functional Studies - NAD+/NADH Assay Kit (ab65348)
    Functional Studies - NAD+/NADH Assay Kit (ab65348)
    NADH Standard calibration curve. Quantification of NAD (diamond) and NADH (open square) following product protocol and using NADH standard provided in the kit. No NADP (triangle) was detected in this reaction.

实验方案

文献

This product has been referenced in:
  • Long YM  et al. Surface ligand controls silver ion release of nanosilver and its antibacterial activity against Escherichia coli. Int J Nanomedicine 12:3193-3206 (2017). Read more (PubMed: 28458540) »
  • Shao D  et al. Glutaredoxin-1 Deficiency Causes Fatty Liver and Dyslipidemia by Inhibiting Sirtuin-1. Antioxid Redox Signal 27:313-327 (2017). Read more (PubMed: 27958883) »

See all 34 Publications for this product

发表研究结果有使用 ab65348？请让我们知道，以便我们可以引用本数据表中的参考文章。

客户评价及客户问答

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106 reviews or Q&A

10
Votes: Highest First

Is the extraction buffer the same for both these kits?

Yes, the extraction buffers from ab65348 and ab65349 are the same.

Could you please provide the range to measure nM for NAD/NADH Assay Kit (ab65348).

I can confirm that for ab65348 the detection range is 1-100 pmoles per well as shown on the STD curve (on-line product datasheet). The detection limit is 1 pmole in the sample volume added into the well.

Can adherent cells be extracted in the plate, rather than removing them by trypsinization or other method and pelleting?

Thank you for your question about how to assay adherent cells. The cells can be lysed by aspirating the medium and adding 400ul of the extraction buffer as we discussed, followed by 2 freeze-thaw cycles. That should be sufficient to lyse all the cells....

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1) How can I avoid the foaminess when the NAD cycling enzyme is redissolved in the NAD cycling buffer (p.7 of protocol)? I used the pipette slowly up and down, but still ˜ 40ul of the 220 ul were lost and I ended up getting way less aliquots than expec...

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1) To prevent extreme foaminess in the tube, do not add the 220ul of the NAD cycling buffer to the enzyme mix. Add a small volume at a time and wait.


2) NAD is much more heat-labile and hence it decomposes at 60C.

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I have purchased the NAD/NADH Assay Kit (ab65348), and I have a question about the protocol. Step 5 says to “let the reaction cycling at room temperature for 1 to 4 hours or longer depending on the reading of OD 450 nm” How do I know when t...

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Please take multiple readings during the 1-4 hr incubation and stop the reaction once the OD of the highest standard reaaches ˜1.9 or starts plateuing.

After adding the stop solution, OD readings will be stable for 48 hours and should be ...

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rat heart tissue samples
400 ug protein in 50 ul per well
OD450 nm read outs:
NADtotal NADH
1.344 1.36
1.355 1.444
baseline: 0.135
but NADH should have smaller values than NADtotal
followed protocol step...

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Thank you for contacting us.
The lab got back to me with the following advice and suggestion:
It seems that 400 µg/well is on the higher side. Please recommend 100-200 µg /well of the protein.
There is also a possibility that the samples c...

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Developer and enzyme mix appearance.

I have confirmed with the lab that the developer is a red powder and the enzyme mix is a clear powder for ab65348.

Is it possible to use more cells, or would you need to use a larger volume of Extraction Buffer if you were to do that?



It is definitely possible to use more cells to get a more concentrated sample. The volume of extraction buffer can be increased slightly, (for example, 500ul for 50,000 cells) but if you scale up the extraction buffer in the same ratio as the...

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We are using NADP/NADPH Assay Kit ab65349 for measuring NADPH in lymphoma
cells currently, but we also want to measure NADH in the same cells.
We are going to order NAD/NADH Assay Kit (ab65348)very soon.
Can we share the cell extracts, whic...

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We recommend using the buffers provided with the specific kits for our assays. So, unfortunately we don’t suggest using the same buffer for both assays (ab65348 and ab65349).

Product code: 65348

Inquiry: Is the NAD/NADH kit working with plant tissue? Thanks,

I am sorry to confirm this kit has not been specifically tested and guaranteed for use on plant samples.

The following references may provide information of preparing plant tissue for NAD and NADP measurement which I hope may be helpful: Read More

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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