CoenzymeⅠNAD(H) Content Assay Kit

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辅酶ⅠNAD(H)含量检测试剂盒

Cat:

NA0601

Assay Type:

Spectrophotometer/Microplate Reader

Brand:

sunlong medical

Specificity:

100T/48S

Storage instructions：

-20℃

Product Overview：

Components:

Acid extract solution：Liquid 25 mL×1. Storage at 4℃.

Alkaline extract solution：Liquid 25 mL×1. Storage at 4℃.

Reagent I：Liquid 5mL×1. Storage at 4℃.

Reagent II：Liquid 1.5mL×1. Storage at 4℃.

Reagent III： Powder×1. Storage at -20℃. Add 1.6 mL of double distilled water before use, mix

thoroughly, store at 4℃ for one week；

Reagent IV：Powder××1. Storage at 4℃. Add 1.9 mL of double distilled water before use, mix thoroughly,

store at 4℃ for one week；

Reagent V：Liquid 0.7mL×1. Storage at 4℃.

Reagent VI：Liquid 15mL×1. Storage at 4℃.

Reagent VII：Self-provided, take 19.2 mL of alcohol and 0.8 mL of distilled water, mix thoroughly.

NAD standard： Powder×1. Storage at -20℃. Add 3 mL of distilled water before use to prepare as

1µmol/mL, then dilute it to 1.25nmol/mL NAD standard solution.

NADH standard：Powder×1. Storage at -20℃. Add 2.8 mL of distilled water before use to prepare as 1

µmol/mL, then dilute it to 1.25 nmol/mL NADH standard solution.

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Unit:

Price: $218

Product PDFs

Datasheet:

Other Information
Citations
Protein Attributes
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Coenzyme I include both reduced and oxidized forms and plays a role in hydrogen transfer in biological

oxidation. Oxidized coenzyme I also called nicotinamide adenine dinucleotide (NAD+) which is the

coenzyme of dehydrogenase. It is an important role in glycolysis, gluconeogenesis, tricarboxylic acid cycle

and respiratory chain. Intermediate product will transfer hydrogen to NAD make it become

NADH(reduced coenzyme I). NADH acts as a carrier for hydrogen and synthesizes ATP by chemosmosis

coupling in the respiratory chain. NADH has important physiological significance in the body. It is closely

related to substance metabolism, energy metabolism, anti-cell aging, anti-oxidation and the occurrence of

some diseases. A decrease in coenzyme I levels in the body can lead to cell damage or death.

Extract the sample of NAD+ and NADH with acidic and alkaline extract solution respectively, NADH

reduces the oxidized Thiazolyl Blue Tetrazolium Blue (MTT) to form formazan by hydrogen transfer from

PMS, formazan has characteristic absorption at 570nm. NAD could be reduced to NADH by alcohol

dehydrogenase. Further, MTT reduction method was used to detect NAD+.

Required but Not Provided:

Microplate reader/Spectrophotometer, desk centrifuge, desk centrifuge, transferpettor, micro glass cuvette/

96 well flat-bottom plate, mortar/ homogenizer, ice and distilled water.

Protocol

I. Extraction of NAD⁺and NADH：

1. Serum

Extraction of NAD⁺：Add 0.5 mL of acid extract solution to 0.1 mL of serum, boiling 5 minutes, ice bath

after cooling. Centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant 200 µL and add equal volume

alkaline extract solution. Mix thoroughly, centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant,

preserve on ice for test.

Extraction of NADH：Add 0.5 mL of alkaline extract solution to 0.1 mL of serum, boiling 5 min, ice bath

after cooling. Centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant 200 µL and add equal volume

acid extract solution. Mix thoroughly, centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant,

preserve on ice for test.

2. Tissue

Extraction of NAD⁺： Add 0.5 mL of acid extract solution to 0.1 g of tissue, grinding on ice, boiling 5

minutes, ice bath after cooling. Centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant 200 µL and

add equal volume alkaline extract solution.Mix thoroughly, centrifuge at 10000 ×g for 10 minutes at 4℃,

take supernatant, preserve on ice for test.

Extraction of NADH：Add 0.5 mL of alkaline extract solution to 0.1 g of tissue, grinding on ice, boiling 5

minutes, ice bath after cooling. Centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant 200 µL and

add equal volume acid extract solution. Mix thoroughly, centrifuge at 10000 ×g for 10 minutes at 4℃, take

supernatant, preserve on ice for test.

3. Cells or microorganism：

Extraction of NAD⁺： Add 0.5 mL of acid extract solution to 5 million cells or germ, ultrasonic

1min(power 20% or 200W, ultrasonic 2s, interval 1s), boiling 5 minutes, ice bath after cooling. Centrifuge

at 10000 ×g for 10 minutes at 4℃, take supernatant 200 µL and add equal volume alkaline extract solution.

Mix thoroughly, centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant, preserve on ice for test.

Extraction of NADH：Add 0.5 mL of alkaline extract solution to 5 million cells or germ, ultrasonic 1min

(power 20% or 200W, ultrasonic 2s, interval 1s) boiling 5 minutes, ice bath after cooling. Centrifuge at

10000 ×g for 10 minutes at 4℃, take supernatant 200 µL and add equal volume acid extract solution. Mix

thoroughly, centrifuge at 10000 ×g for 10 minutes at 4℃, take supernatant, preserve on ice for test.

II.

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