NAD/NADH Quantitation Kit

Details for Product No. ABIN411692
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Target Name (Antigen)
Methode Type Competition ELISA
Application
Biochemical Assay (BCA)
Pubmed 6 references available
Quantity 100 tests
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Catalog No. ABIN411692
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Sample Type Cell and Tissue Culture Lysate, Urine, Plasma, Serum, Biological Fluids
Specificity Assay of nicotinamide nucleotides is of continual interest in the studies of energy transforming and redox state of cells or tissues. NADH/NAD Quantification Kit provides a convenient tool for sensitive detection of the intracellular nucleotides: NADH, NAD and their ratio. 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 NADH and NAD, 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.
Components NADH/NAD Extraction Buffer, NAD Cycling Buffer, NAD Cycling Enzyme Mix, NADH Developer, Stop Solution, NADH Standard (MW:763)
Protocol A. Reagent Reconstitution and General Consideration: Reconstitute NAD Cycling Enzyme Mix with 220 µL NAD Cycling Buffer. Reconstitute NADH developer with 1.2 mL of ddH2O. Pipette up and down several times to completely dissolve the pellet into solution (don't vortex). Aliquot enough NAD Cycling Enzyme mix (2 µL per assay) for the number of assays to be performed in each experiment and freeze the stock solution immediately at -70 °C for future use. The enzymes are stable for up to 2 months at -70 °C after reconstitution. Reconstitute NADH standard with 200 µL pure DMSO to generate 1 nM/µL NADH standard solution. Ensure that the NAD Cycling Buffer is at room temperature before use. Keep other enzymes on ice during the assay and protect from light. B. Sample Preparation:
1. For cell samples*, wash cells with cold PBS. Pellet 2X 10 5 cells for each assay in a micro-centrifuge tube (2000 rpm for 5 min). Extract the cells with 400 µL of NADH/NAD Extraction Buffer by freeze/thaw two cycles (20 min on dry-ice, then 10 min at room temperature), or homogenization. Vertex the extraction for 10 sec. Spin the sample at 14000 rpm for 5 min. Transfer the extracted NADH/NAD supernatant into a labeled tube.
2. For tissue samples*, weight approx. 20 mg tissue, wash with cold PBS, homogenize with 400 µL of NADH/NAD Extraction Buffer in a micro-centrifuge tube. Spin the sample at 14000 rpm for 5 min. Transfer the extracted NADH/NAD supernatant into a new tube. Note: Cell or tissue lysates may contain enzymes that consume NADH rapidly. We suggest to remove these enzymes by filtering the samples through 10 Kd molecular weight cut off filters before performing the assay. C. NADH/NAD
1. Standard Curve: Dilute 10 µL of the 1 nM/µL NADH standard with 990 µL NADH/NAD Extraction Buffer to generate 10 pM/µL standard NADH (Note: diluted NADH solution is unstable, must be used within 4 hours). Add 0, 2, 4, 6, 8, 10 µL of the diluted NADH standard into labeled 96-well plate in duplicate to generate 0, 20, 40, 60, 80, 100 pM/well standard. Make the final volume to 50 µL with NADH/NAD extraction buffer. Samples: To detect total NADt (NADH and NAD), transfer 50 µL of extracted samples into labeled 96-well plate in duplicates. (Note: We recommend performing several different sample dilutions with the Extraction Buffer to ensure the readings fall in the standard curve range). To detect NADH, NAD needs to be decomposed before the reaction. To decompose NAD, aliquot 200 µL the extracted samples into eppendorf tubes. Heat to 60 °C for 30 min in a water bath or a heating block. Under the condition, all NAD will be decomposed, while NADH will still be intact. Cool samples on ice. Quick spin the samples to remove precipitates if precipitation occurs.
Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Product cited in: Hammill, Welles, Carson: "The gel microdrop secretion assay: Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells." in: Cytotechnology, Vol. 34, Issue 1-2, pp. 27-37, 2008 (PubMed).

Pang, Gong, Xi et al.: "Poly(ADP-ribose) polymerase 1 is involved in glucose toxicity through SIRT1 modulation in HepG2 hepatocytes." in: Journal of cellular biochemistry, Vol. 112, Issue 1, pp. 299-306, 2010 (PubMed).

Kulkarni, Donepudi, Xu et al.: "Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human." in: Antioxidants & redox signaling, 2013 (PubMed).

Oppenheimer, Kumar, Meir et al.: "Set7/9 impacts COL2A1 expression through binding and repression of SirT1 histone deacetylation." in: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2013 (PubMed).

General Wang, Xu, Guan et al.: "Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway." in: Annals of neurology, 2011 (PubMed).

Dvir-Ginzberg, Gagarina, Lee et al.: "TNFα-mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes." in: Arthritis and rheumatism, 2011 (PubMed).

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