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

Cat. No.: N1501270

GRADE & PURITY BioReagent

Overview

NAD Kinase (NADK, EC 2.7.1.23) is widely present in animals, plants, microorganisms, and cultured cells. It is the only known enzyme that catalyzes the phosphorylation of NAD⁺ to NADP⁺ in vivo. It can utilize ATP or inorganic polyphosphate [poly(P)] as a phosphoryl donor to catalyze the phosphorylation of NAD(H), generating NADP(H). Therefore, NADK plays a crucial role in synthesizing NADP(H) and regulating the balance between NAD(H) and NADP(H).

Assay Principle
NADK catalyzes the phosphorylation of NAD⁺ to generate NADP⁺. The generated NADP⁺ is then reduced to NADPH by Glucose-6-Phosphate Dehydrogenase (G6PDH). The rate of increase in NADPH, measured by the rise in absorbance at 340 nm, reflects the activity of NADK.

Component	50T	Storage
Extraction Buffer	50 mL	2-8℃
Reagent 1	25 mL	2-8℃
Reagent 2	50 mL	2-8℃
Reagent 3	1EA	-20℃
Reagent 4	1EA	-20℃

Required Materials and Equipment (Not Provided)
UV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.

Sample Preparation

1.Bacteria, Cells, or Tissues:

Bacteria or Cultured Cells: Collect cells by centrifugation and discard the supernatant. Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells). Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice.

Tissues: Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer). Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice.

2.Serum (or Plasma) Samples: Assay directly.

Assay Procedure:

1.Preheat the spectrophotometer for at least 30 min. Set wavelength to 340 nm. Zero with distilled water.

2.Pre-warm Reagent 1 and Reagent 2 at 37°C (for mammalian samples) or 25°C (for other species) for at least 15 min.

3.Working Solution I Preparation: Add 12 mL of Reagent 1 to the contents of Reagent 3. Mix thoroughly. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

Working Solution II Preparation: Add 45 mL of Reagent 2 to the contents of Reagent 4. Mix thoroughly. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

4.Assay Setup:

Reagent	Test Tube (μL)	Control Tube (μL)
Sample	100	100
Working Solution I	400
Reagent 1		400

Mix thoroughly. Incubate at 37°C (mammalian) or 25°C (other species) for 15 min.

Immediately boil for 2 min (tighten caps to prevent evaporation).

Cool on ice.

Centrifuge at 10,000 g, 25°C for 10 min. Collect the supernatant.

5.Detection:

Reagent	Volume (μL)
Supernatant (from step 4)	200
Working Solution II	800

• Add reagents to a new tube or cuvette. Mix thoroughly after addition.

• Let the reaction stand at room temperature for 15 min.

• Measure the absorbance at 340 nm.

• Calculate ΔA = A_Test - A_Control.

NADK Activity Calculation:

General Parameters:

• V_Total (Total reaction volume for detection step) = 5 × 10⁻⁴ L (0.5 mL = 500 μL)

• ε (NADPH molar extinction coefficient) = 6.22 × 10³ L/mol/cm

• d (Cuvette light path) = 1 cm

• V_Sample (Sample volume in initial reaction) = 0.1 mL (100 μL)

• V_{Sample Total} (Total extraction volume) = 1 mL

• T (Reaction time for NADK enzyme step) = 15 min

• Cpr (Sample protein concentration, mg/mL)

• W (Sample mass, g)

• 500 (Cell/Bacteria count in millions for example calculation: 5 million)

1. For Serum (Plasma):

• Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per ml of serum.

• Calculation:
  NADK Activity (nmol/min/ml) = [ΔA × V_Total ÷ (ε × d) × 10⁹] ÷ V_Sample ÷ T
  Simplified Formula: NADK (nmol/min/ml) = 53.59 × ΔA

2. For Tissues, Bacteria, or Cells:

• a. Based on Sample Protein Concentration:

  • Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per mg of protein.

  • Calculation:
    NADK Activity (nmol/min/mg prot) = [ΔA × V_Total ÷ (ε × d) × 10⁹] ÷ (V_Sample × Cpr) ÷ T
    Simplified Formula: NADK (nmol/min/mg prot) = 53.59 × ΔA ÷ Cpr

• b. Based on Sample Fresh Weight:

  • Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per gram of fresh tissue.

  • Calculation:
    NADK Activity (nmol/min/g fresh weight) = [ΔA × V_Total ÷ (ε × d) × 10⁹] ÷ (W × V_Sample / V_{Sample Total}) ÷ T
    Simplified Formula: NADK (nmol/min/g fresh weight) = 53.59 × ΔA ÷ W

• c. Based on Bacterial or Cell Density:

  • Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per 10⁴ cells.

  • Calculation (example for 5 million cells in 1 ml extract):
    NADK Activity (nmol/min/10⁴ cell) = [ΔA × V_Total ÷ (ε × d) × 10⁹] ÷ (500 × V_Sample / V_{Sample Total}) ÷ T
    Simplified Formula: NADK (nmol/min/10⁴ cell) = 0.107 × ΔA

Precautions
Before formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity.

Storage and Shipping

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 6 months under corresponding storage conditions.