Assays for Determination of Cellular and Mitochondrial NAD + and NADH Content

Assays for Determination of Cellular and Mitochondrial NAD + and NADH Content
NAD+ is a redox cofactor important to the right functioning of a wide range of essential metabolic pathways, together with key steps in mitochondrial vitality metabolism. As well as, it serves as a signaling substrate for enzymes reminiscent of sirtuins and the poly-ADP ribosyl-polymerase household of enzymes.
Sirtuins, that are NAD+-dependent protein deacylases, harness modifications in mobile NAD+ concentrations to provide modifications in protein acylation standing, thereby affecting downstream features together with vitality metabolism, stress resistance, and cell survival.
Thus, the provision of NAD+ in cells, or in particular organelles such because the mitochondrion, regulates downstream signaling and key organic processes. This idea has pushed a necessity for researchers to simply and exactly measure NAD+ concentrations in organic samples.
We herein describe a number of protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments reminiscent of mitochondria. These protocols embody a biking assay that may shortly measure NAD+ or NADH ranges utilizing a plate reader outfitted with fluorescence measurement capabilities.
This plate assay depends solely upon commercially out there supplies along with the organic samples of curiosity. As well as, we describe a protocol using steady isotope-labeled NAD+ as an inside customary to find out organic NAD+ content material by isotope-dilution strategies. This technique requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to acquire quantification utilizing HPLC and mass spectrometry.

A novel light-controlled colorimetric detection assay for nitroreductase primarily based on p-aminophenol-catalyzed and NADH-mediated synthesis of silver nanoparticles

A novel and environment friendly light-controlled colorimetric assay for the quantification and detection of nitroreductase (NTR) was constructed primarily based on p-aminophenol (pAP)-catalyzed and nicotinamide adenine dinucleotide (NADH)-mediated technology of AgNPs.
Because of the hydrolysis of p-nitrophenol by NTR within the presence of NADH, the hydrolysis product can be utilized as a catalyst to catalyze the discount of Ag+ by NADH beneath the sunshine. Because the focus of NTR will increase, the worth of absorbance at ca. 400 nm (A400) decreases and the colour of the answer turns from brown to vivid yellow.
A linear correlation was obtained between A400 and the NTR focus within the vary from 1-50 μg mL-1 and the restrict of detection (LOD) is 0.27 μg mL-1.
The detection system doesn’t reply to different widespread organic molecules because of the specificity of enzymes and the impact of the nitroreductase inhibitor on the NTR exercise was additionally examined.
Lastly, we utilized the assay to find out NTR in human serum samples by spiking totally different concentrations of NTR with a restoration of 85.2%-92.5%.

Identification of proteins able to metallic discount from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 utilizing an NADH-based exercise assay.

Understanding of microbial metallic discount relies nearly solely on research of Gram-negative organisms. On this research, we deal with Desulfotomaculum reducens MI-1, a Gram-positive metallic reducer whose genome lacks genes with similarity to any characterised metallic reductase.
Utilizing non-denaturing separations and mass spectrometry identification, together with a colorimetric display screen for chelated Fe(III)-NTA discount with NADH as electron donor, we’ve got recognized proteins from the D. reducens proteome not beforehand characterised as iron reductases.
Their perform was confirmed by heterologous expression in Escherichia coli. Moreover, we present that these proteins have the aptitude to scale back soluble Cr(VI) and U(VI) with NADH as electron donor. The proteins recognized are NADH : flavin oxidoreductase (Dred_2421) and a protein complicated composed of oxidoreductase flavin adenine dinucleotide/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686).
Dred_2421 was recognized within the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 had been recognized in each the soluble in addition to the insoluble protein fraction, suggesting a sort of membrane affiliation, though PSORTb predicts each proteins are cytoplasmic. This research is the primary purposeful proteomic evaluation of D. reducens and one of many first analyses of metallic and radionuclide discount in an environmentally related Gram-positive bacterium.
Assays for Determination of Cellular and Mitochondrial NAD + and NADH Content

NADH-driven poly-3-hydroxybutyrate accumulation in Escherichia coli: Knowledge from enzymatic assays and oxygen-limited steady cultures

Biosynthesis of poly-3-hydroxybutyrate (PHB) as a fermentation product allows the coupling of progress and product technology. Furthermore, the discount of oxygen provide ought to cut back operative value and enhance product yield.
Era of PHB as a fermentation product relies on the in vivo exercise of an NADH-preferring acetoacetyl-CoA reductase.
Proof of this idea requires
(i) quantification of the cofactor desire, in physiologically related circumstances, of a putative NADH-preferring acetoacetyl-CoA reductase and
(ii) verification of PHB accumulation utilizing an NADH-preferring acetoacetyl-CoA reductase in a species naturally incapable of doing so, for instance, Escherichia coli.
This dataset comprises kinetic knowledge obtained by spectrophotometry and knowledge from a steady tradition of an engineered E. coli pressure accumulating PHB beneath oxygen-limiting circumstances.
On this dataset it’s attainable to search out
(1) enzyme stability assays;
(2) preliminary charges and progress curves from reactions catalyzed by two acetoacetyl-CoA reductases;
(3) estimations of the relative use of NADH and NADPH by two acetoacetyl-CoA reductases;
(4) estimations of the flux capability of the response catalyzed by an acetoacetyl-CoA reductase;
(5) biomass composition of an engineered E. coli pressure remodeled with a plasmid;
(6) calculation of reconciled particular charges of this engineered pressure rising on sucrose as the only real carbon supply beneath oxygen limitation and
(7) metabolic fluxes distributions throughout the steady progress of this engineered pressure.
As a result of a comparatively small variety of acetoacetyl-CoA reductases have been kinetically characterised, knowledge and scripts right here offered may very well be helpful for additional kinetic characterizations.
Furthermore, the process described to estimate biomass composition may very well be fascinating to estimate plasmid and protein burden in different strains. Software of knowledge reconciliation to fermentations ought to assist to acquire particular charges in step with the precept of mass and electron conservation.
All of the required knowledge and scripts to carry out these analyses are deposited in a Mendeley Knowledge repository. This text was co-submitted with the manuscript entitled “An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichiasia. coli”.

The next sensitivity and effectivity of widespread primer multiplex PCR assay in identification of meat origin utilizing NADH dehydrogenase subunit Four gene.

A Widespread Primer Multiplex PCR (CP-M-PCR) was developed to detect meat origin of 4 teams of animal (pig, ruminant, avian and rabbit). This technique demonstrated increased sensitivity and effectivity than the traditional multiplex PCR.
On this method, a standard ahead primer was designed within the 5′ finish of a homologous area of mitochondrial NADH dehyrogenase subunit 4 (Nad 4) gene sequences of all of the animal teams. Particular adapter reverse primers had been designed by including an adapter sequence on the 5′ finish.
The identical adapter sequence was used because the widespread adapter reverse primer. The primers generated particular fragments of 267, 370, 504, and 548 bp lengths for pig, ruminant, avian and rabbit meats, respectively. The usage of adapter sequence on the 5′ finish of the widespread adapter reverse primers elevated the effectivity of the amplification and the appliance of a standard ahead primer solved the complexity in multiplex PCR system.
Bands of particular amplification will be detected within the PCR assays containing as little as 10(-6) μM of adapter reverse primer. This end result indicated that the sensitivity was tremendously elevated as in comparison with the traditional multiplex PCR (10(-3) μM).

Amplite™ Colorimetric NADH Assay Kit

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NADH Oxidase Activity Assay Kit (Fluorometric)

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Amplite™ Fluorimetric NADH Assay Kit *Red Fluorescence*

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Amplite™ Colorimetric NAD/NADH Ratio Assay Kit

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EZScreen? NAD+/NADH Colorimetric Assay Kit (384-well)

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Amplite™ Colorimetric Total NAD and NADH Assay Kit

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Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*

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Amplite™ Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*

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Amplite™ Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity*

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NADH Na2

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  • 1 g
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NADH, disodium salt

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NADH Quantitation Kit

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Description: NADH Quantitation Kit for use in the research laboratory

XTT CELL VIABILITY ASSAY KIT (1000 ASSAY)

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Description: Minimum order quantity: 1 unit of 1KIT

Endothelial Tube Formation Assay (In Vitro Angiogenesis Assay)

CBA-200 50 assays
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Description: For angiogenesis to occur, endothelial cells must escape their stable location and break through the basement membrane. Cells migrate toward an angiogenic stimulus that may be released from nearby tumor cells. These cells proliferate to form new blood vessels. Our Endothelial Tube Formation Assay (In Vitro Angiogenesis) provides an easy, robust system to assess angiogenesis in vitro. The ECM gel matrix very closely resembles an in vivo environment.

Lactose Assay Kit

MET-5001 100 assays
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Description: The Lactose Assay Kit measures total lactose in milk based food products or biological samples such as blood or urine. Lactose is cleaved into glucose and galactose. Glucose is then oxidized, yielding hydrogen peroxide and D-gluconic acid. The hydrogen peroxide is detected by a fluorometric probe.

Bilirubin Assay Kit

MET-5010 200 assays
EUR 479
Description: Bilirubin, a byproduct of heme breakdown, can exist conjugated to glucuronic acid (direct) and as unconjugated (indirect). The unconjugated form is found in the blood bound to albumin and is transported to the liver. Bilirubin becomes conjugated to glucuronic acid in the liver, making it more soluble and allowing for excretion into bile. High levels of bilirubin have been correlated with jaundice and Gilbert?s syndrome while low levels have been associated with cardiovascular disease and diabetes mellitus.

Pyruvate Assay Kit

MET-5029 100 assays
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Description: Our Pyruvate Assay Kit measures pyruvate in biological samples. First, pyruvate is oxidized by pyruvate oxidase, producing hydrogen peroxide. The hydrogen peroxide is then detected at ex. 530-570 nm/em. 590-600 nm using a specific fluorometric probe. Pyruvate levels in unknown samples are determined based on the provided pyruvate standard curve.

Glycine Assay Kit

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Histamine Assay Kit

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Description: Histamine is naturally occurring in food, with high concentrations associated with spoiled and fermented foods. Exposure to high levels of histamine through the ingestion of food can cause symptoms similar to an allergic response. Our Histamine Assay Kit detects total histamine from food samples using a colorimetric probe. Reduction of the probe yields color development proportional the histamine levels in the sample. Absorbance at 450nm is read after a one hour incubation at 37C and histamine levels are calculated based on a histamine standard curve.

Glucose Assay Kit

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NADPase Assay Kit

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CP-M-PCR detection restrict of the DNA samples was 0.1 ng for the 4 teams of meats. CP-M-PCR has enormously improved the sensitivity and effectivity of the PCR system for a extra dependable and correct final result than typical multiplex PCR system.

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