Nadp nadph quantitation kit

Manufactured by Merck Group

Sourced in United States

The NADP/NADPH Quantitation Kit is a laboratory tool designed to quantify the levels of NADP (nicotinamide adenine dinucleotide phosphate) and NADPH (the reduced form of NADP) in biological samples. The kit provides a colorimetric assay that enables the measurement of these important cellular cofactors.

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Lab products found in correlation

Multiskan go, by Thermo Fisher Scientific (2 mentions) H2dcfda, by Merck Group (1 mentions) Hpx 87h column, by Aminex (1 mentions) Uplc beh amide column, by Waters Corporation (1 mentions) Uhplc system, by Agilent Technologies (1 mentions) Nad nadh quantitation kit, by Merck Group (1 mentions) Tripletof 6600 q tof, by AB Sciex (1 mentions) Ab93349, by Abcam (1 mentions) Nad nadh quantification kit, by Merck Group (1 mentions) Perhexiline, by Merck Group (1 mentions) Nad nadh assay kit, by Abcam (1 mentions) Celltiter glo luminescent cell viability assay, by Promega (1 mentions) Nad nadh quantitation colorimetric kit, by Abcam (1 mentions) Imarktm, by Bio-Rad (1 mentions) Complex 1 enzyme activity microplate assay kit, by Abcam (1 mentions) Deproteinization sample preparation kit, by Abcam (1 mentions) Atp colorimetric fluorometric assay kit, by Abcam (1 mentions) Bradford reagent, by Merck Group (1 mentions) Starch colorimetric fluorometric assay kit, by Abcam (1 mentions) Spectrophotometric multi well plate reader, by Tecan (1 mentions)

13 protocols using nadp nadph quantitation kit

Quantifying Yeast Oxidative Stress

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ROS levels in yeast cells were assayed with 2,7′-dichlorodihydrofluorescein diacetate (Sigma-Aldrich H2DCF-DA). Briefly, strains were incubated in YPD broth for 4 h at 40°C, and then H2DCF-DA was added to a final concentration of 10 μM and incubated for 30 min in the dark at 40°C. Yeast cells were collected by centrifuge and washed three times with phosphate buffered saline (PBS; 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, and 8 mM K₂HPO₄ at pH 7.2). Fluorescence intensity was measured by spectrophotometry with excitation of 488 nm and emission of 525 nm.
NADP⁺ and NADPH were extracted and measured using the NADP⁺/NADPH quantitation kit (Sigma-Aldrich) as described by Zhang et al. (2016) (link).

Song L., Shi J.Y., Duan S.F., Han D.Y., Li K., Zhang R.P., He P.Y., Han P.J., Wang Q.M, & Bai F.Y. (2021). Improved redox homeostasis owing to the up-regulation of one-carbon metabolism and related pathways is crucial for yeast heterosis at high temperature. Genome Research, 31(4), 622-634.

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NADPH Quantification Using Sigma-Aldrich Kit

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NADPH was measured using an NADP/NADPH Quantitation Kit (Sigma-Aldrich, Cat. No. MAK038) according to the manufacturer's instructions [102 (link)].

Riz I., Hawley T.S., Marsal J.W, & Hawley R.G. (2016). Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming. Oncotarget, 7(41), 66360-66385.

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Metabolite Quantification by LC-MS/MS

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Cell growth was determined by measuring the OD₆₀₀ using a Beckman Coulter DU370 spectrophotometer. Methanol concentrations were measured using an Agilent 1290 Infinity System (Santa Clara, CA, USA) equipped with an Aminex HPX-87H column. l-Lysine was determined using SBA-40E immobilized enzyme biosensor (Shandong, China) [21 (link)]. NADH and NADPH were assayed by means of the NAD/NADH quantitation Kit and the NADP/NADPH quantitation Kit (Sigma-Aldrich).
LC–MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) equipped with a UPLC BEH Amide column (1.7 μm, 2.1*100 mm, Waters) coupled to Triple TOF 6600 (Q-TOF, AB Sciex). The mobile phase consisted of 25 mM NH₄OAc and 25 mM NH₄OH in water (pH = 9.75) (A), and acetonitrile (B) was carried with elution gradient as follows: 0 min-95% B, 0.5 min-95% B, 7 min-65% B, 8 min-40% B, 9 min-40% B, 9.1 min-95%, and B, 12 min-95% B, which was delivered at 0.5 mL/min. The injection volume was 1 μL. The Triple TOF mass spectrometer was used to assess its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during a LC/MS experiment. ESI source conditions were set as follows: Ion source gas 1 as 60, Ion source gas 2 as 60, Curtain gas as 35, source temperature 550 °C, Ion Spray Voltage Floating (ISVF) 5500 V or − 4500 V in positive or negative modes, respectively.

Wang X., Wang X., Lu X., Ma C., Chen K, & Ouyang P. (2019). Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli. Biotechnology for Biofuels, 12, 17.

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Quantifying NADPH and NADP+ Levels

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Levels of NADPH and NADP+ were measured using a NADP/NADPH Quantitation Kit (Sigma-Aldrich, USA) following the manufacturer's instructions. In brief, the cells were treated with EBSS and 10 μM SBI-0206965. The cells were then treated with NADP/NADPH extraction buffer and the NADPH developer was added. The reaction mixture was incubated at room temperature for 1 h. The samples were analysed at an absorbance at 450 nm using a spectrophotometer.

Lu J., Zhu L., Zheng L.P., Cui Q., Zhu H.H., Zhao H., Shen Z.J., Dong H.Y., Chen S.S., Wu W.Z, & Tan J.M. (2018). Overexpression of ULK1 Represents a Potential Diagnostic Marker for Clear Cell Renal Carcinoma and the Antitumor Effects of SBI-0206965. EBioMedicine, 34, 85-93.

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Niacin Index Quantification in RBCs

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Niacin index is calculated as NAD/NADP X 100. RBCs were lysed as described in the Methods outlined in the following kit manuals. To obtain the niacin index (NAD/NADP), the NAD/NADH quantification kit (Sigma-Aldrich Cat # MAK037) and NADP/NADPH quantitation kit (Sigma-Aldrich Cat # MAK038) were used in tandem. 10 µl of RBC samples were used for each kit according to manufacturer’s guidelines in a 96-well plate with included standards. Samples were filtered using a 10 kda MW cut off spin filter (ABCAM Cat # ab93349) spun at 20,000×g for 30 minutes. All colorimetric detection were accomplished using spectrophotometer absorbance at A = 450 nm. The niacin index and the NAD/NADH ratio were then calculated for each sample.

Wakade C., Chong R., Bradley E., Thomas B, & Morgan J. (2014). Upregulation of GPR109A in Parkinson’s Disease. PLoS ONE, 9(10), e109818.

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NADP/NADPH Quantitation Assay

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The same samples that were prepared for NAD cycling assays were used for NADP cycling assays. 50 μL of each sample was used as input for the NADP/NADPH Quantitation Kit (Sigma-Aldrich, MAK038-1KT) according to the instructions provided by the manufacturer.

Koopal B., Potocnik A., Mutte S.K., Aparicio-Maldonado C., Lindhoud S., Vervoort J.J., Brouns S.J, & Swarts D.C. (2022). Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell, 185(9), 1471-1486.e19.

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Metabolic Profiling of Perhexiline-Treated Cells

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The indicated EOC cells were seeded in 96-well plates (2 × 10⁴ cells) and treated with 10 μM Perhexiline for 24 h. The ATP, NADH and NADPH levels were determined after treatment with Perhexiline (Sigma-Aldrich) by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), NAD/NADH Assay kit (Abcam, ab65348) and NADP/NADPH Quantitation Kit (Sigma-Aldrich, MAK038-1KT) according to the manufacturer's instructions, respectively.

Zhu J., Wu G., Song L., Cao L., Tan Z., Tang M., Li Z., Shi D., Zhang S, & Li J. (2019). NKX2-8 deletion-induced reprogramming of fatty acid metabolism confers chemoresistance in epithelial ovarian cancer. EBioMedicine, 43, 238-252.

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Quantifying NADPH and NADP+ Levels

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Levels of NADPH and NADP+ were measured using a NADP/NADPH Quantitation Kit (Sigma-Aldrich, USA) following the manufacturer’s instructions. In brief, ABAT, ALDH6A1 stable overexpressed or control ACHN and 786-O cells were treated with NADP/NADPH extraction buffer and the NADPH developer was added. The reaction mixture was incubated at room temperature for 1 h. The samples were analysed at an absorbance at 450 nm using a spectrophotometer (Multiskan GO, Thermo Scientific).

Lu J., Chen Z., Zhao H., Dong H., Zhu L., Zhang Y., Wang J., Zhu H., Cui Q., Qi C., Wang S., Chen S, & Shao J. (2020). ABAT and ALDH6A1, regulated by transcription factor HNF4A, suppress tumorigenic capability in clear cell renal cell carcinoma. Journal of Translational Medicine, 18, 101.

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Quantification of NADPH/NADP Levels

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Reduced and oxidized fractions of nicotinamide adenine dinucleotide phosphates (NADPH/NADP) were measured using the NADP/NADPH Quantitation kit (Sigma-Aldrich, USA) adhering to the manufacturer’s instructions. To detect NADPH, NADP was decomposed by heating sample aliquots at 60 °C for 30 min.

Homolak J., Babic Perhoc A., Knezovic A., Osmanovic Barilar J., Virag D., Joja M, & Salkovic-Petrisic M. (2021). The Effect of Acute Oral Galactose Administration on the Redox System of the Rat Small Intestine. Antioxidants, 11(1), 37.

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NADP/NADPH Quantitation in MDCK Cells

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NADP/NADPH quantitation assays were performed in MDCK cell line following the instruction of NADP/NADPH quantitation kit (Sigma, St. Louis, MO). The transfected MDCK cells were wash with cold PBS, digested with trypsin and pelleted 4 × 10⁶ cells for each assay by centrifuging at 2000 rpm for 5 min. The pelleted cells were extracted with 800 μL of NADP/NADPH Extraction Buffer followed by 10 min standing on ice. The samples were centrifuged for 10 min at 10,000 ×g to remove insoluble material. Then 50 μL supernatant was transferred into a labeled 96 well plate in duplicate. The Master Reaction Mix was set up following the manual and was added into each sample followed by incubation of 5 min at room temperature. 10 μL of NADPH developer was added and incubated at room temperature for 1–4 h according to the color. The absorbance was measure at 450 nm. 10 μL Stop Solution was added into the reactions and the color was stable within 48 h. The results were calculated according to the instructions.

Lyu Z., Mao Z., Li Q., Xia Y., Liu Y., He Q., Wang Y., Zhao H., Lu Z, & Zhou Q. (2018). PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules. EBioMedicine, 38, 178-190.

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