NAD Kinase (NADK) Activity Assay Kit (UV Micro Method) - BioReagent, high purity

Cat. No.: N1501164

GRADE & PURITY BioReagent

Overview

  NAD kinase (NADK, EC 2.7.1.23) is widely present in animals, plants, microorganisms, and cells. It is currently the only enzyme discovered in organisms that can catalyze the phosphorylation of NAD⁺ to generate NADP⁺. It catalyzes the phosphorylation of NAD(H) using ATP or inorganic polyphosphate [poly(P)] as the phosphoryl donor to produce NADP(H). Therefore, NADK plays a crucial role in synthesizing NADP(H) and regulating the balance between NAD(H) and NADP(H).

Detection Principle: In the assay, NADK present in the sample catalyzes the phosphorylation of NAD⁺ to generate NADP⁺. NADP⁺ can then be reduced to NADPH by glucose-6-phosphate dehydrogenase. NADPH has a characteristic absorption peak at 340 nm. The NADK activity is reflected by measuring the rate of increase in NADPH (the change in absorbance) at 340 nm.

Applicable Samples: Serum (plasma), animal/plant tissues, cells, bacteria.

N1501164	Component	48T	96T	Storage
N1501164A	Extraction Buffer	60 mL	60 mL×2	2-8℃
N1501164B	Assay Buffer Ⅰ	6 mL	12 mL	2-8℃
N1501164C	Assay Buffer Ⅱ	15 mL	30 mL	2-8℃
N1501164D	Substrate Mix	1EA	1EA	-20℃. Store in the dark.
N1501164E	Enzyme Mix	1EA	1EA	-20℃. Store in the dark.

Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

User-Provided Instruments and Reagents

• Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)

• 96-well UV plate or micro quartz cuvettes, Adjustable pipettes and tips

• Low-temperature centrifuge, Incubator, Water bath

• Deionized water

• Homogenizer (for tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Assay Buffer I	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Assay Buffer II	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Working Reagent I	Prepare before use: Add 3 mL (48T) / 6 mL (96T) of Assay Buffer I to the Substrate Mix vial. Mix thoroughly.	Unused reagent can be aliquoted and stored at -20°C protected from light for one week. Avoid repeated freeze-thaw cycles.
Working Reagent II	Prepare before use: Add 12.6 mL (48T) / 25.2 mL (96T) of Assay Buffer II to the Enzyme Mix vial. Mix thoroughly.	Unused reagent can be aliquoted and stored at -20°C protected from light for one week. Avoid repeated freeze-thaw cycles.
Substrate Mix		Must be kept on ice during the assay procedure.
Enzyme Mix		Must be kept on ice during the assay procedure.

Note: The temperature of the assay reaction affects the results. Please control it at 25°C (for general species) or 37°C (for mammals).  

2. Sample Preparation

2.1 Plasma and Serum Samples: Can be detected directly.
2.2 Tissue Samples: Rinse the tissue with cold PBS to remove blood from the surface. Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.3 Cell and Bacterial Samples: Collect 5 million bacteria or cells into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.4 Plant Samples: Rinse the plant sample with cold PBS to remove surface impurities. Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
Note: For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.

3. Assay Steps
3.1 Instrument Preparation: Preheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. For UV spectrophotometers, zero the instrument with deionized water.
3.2 Pre-incubate Assay Buffer I in a water bath at 37°C (for mammals) or 25°C (for other species) for at least 15 minutes.
3.3 Set up Test and Control tubes. Add reagents in EP tubes as follows:

Reagent	Control Tube (µL)	Test Tube (µL)
Sample	20	20
Working Reagent I	0	80
Assay Buffer I	80	0

Mix well and incubate in a water bath at 37°C (for mammals) or 25°C (for other species) for 15 minutes. Immediately boil for 2 minutes (cap tightly to prevent moisture loss). Cool in an ice bath. Centrifuge at 10,000 g, room temperature for 10 minutes. 

3.4 Transfer 40 µL of the supernatant from each tube to a 96-well UV plate or micro quartz cuvette. Add 160 µL of Working Reagent II to each, and mix rapidly. 

3.5 After adding reagents and mixing, let stand at room temperature for 15 minutes. Measure the absorbance at 340 nm. Calculate ΔA = A _test - A _control .

Note: A Control tube is required for each sample. It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If the sample ΔA is less than 0.01, appropriately increase the sample amount. If the sample ΔA is greater than 2.0, dilute the sample further with Extraction Buffer and multiply the final result by the dilution factor. 

4. Result Calculation We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas. Calculation formulas for using a 96-well UV plate: 

4.1 NADK Activity in Serum (Plasma): Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per mL of serum (plasma). 

Formula: 

NADK (nmol/min/mL) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ V _sample ÷ T = 107.18 × ΔA 

4.2 NADK Activity in Tissues, Bacteria, or Cells: 

 (1) Based on sample protein concentration:

Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per mg of protein. 

 Formula: 

NADK (nmol/min/mg prot) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ (V _sample × Cpr) ÷ T = 107.18 × ΔA ÷ Cpr 

(2) Based on sample fresh weight: 

 Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per gram of fresh tissue. 

 Formula: 

NADK (nmol/min/g fresh weight) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ (W × V _sample ÷ V _{total extract} ) ÷ T = 107.18 × ΔA ÷ W 

(3) Based on bacterial or cell density: 

 Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per 10⁴ bacteria or cells. 

 Formula: 

NADK (nmol/min/10⁴) = [ΔA × V _{total reaction} ÷ (ε × d) × 10⁹] ÷ (500 × V _sample ÷ V _{total extract} ) ÷ T = 0.214 × ΔA 

Parameter Description: 

 V _{total reaction} : Total reaction volume, 1 × 10⁻⁴ L 

 ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/mol/cm 

 d: Light path of the 96-well UV plate, 0.5 cm 

 10⁹: Conversion factor (1 mol = 10⁹ nmol) 

 V _sample : Volume of sample added, 0.02 mL 

 V _{total extract} : Volume of Extraction Buffer added, 1 mL 

 T: Reaction time, 15 min Cpr: Sample protein concentration, mg/mL 

 W: Sample mass, g 

 500: Total number of bacteria or cells, 5 million 

 Calculation formulas for using micro quartz cuvettes: 

Adjust the light path in the formulas above from d=0.5 cm to d=1 cm.

Precautions

1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

3. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.

4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.

5. This product is for scientific research use only. Not intended for clinical diagnosis. 

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.