Glucose-6-Phosphate Dehydrogenase (G6PDH) Activity Assay Kit (UV Micro Method) - BioReagent

Cat. No.: G1506775

GRADE & PURITY BioReagent

Synonyms

Glucose-6-Phosphate Dehydrogenase (G-6-PD) Activity Assay Kit

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Application

Cell Metabolism, Enzyme activity assay

Overview

  Glucose-6-Phosphate Dehydrogenase (G6PD or G6PDH) (EC 1.1.1.49) is a cytosolic enzyme that catalyzes a chemical reaction. This enzyme participates in the pentose phosphate pathway, a metabolic pathway that supplies reducing power to cells (such as red blood cells) by maintaining NADPH levels. NADPH, in turn, maintains glutathione levels in these cells, protecting red blood cells from oxidative damage caused by compounds like hydrogen peroxide.

  Assay Principle:G6PDH present in the sample converts NADP⁺ to NADPH. NADPH has a characteristic absorption peak at 340 nm. The absorbance value of NADPH is proportional to the G6PDH activity present in the sample.

  Applicable Samples: Serum (Plasma), Animal/Plant Tissues, Cells, Bacteria

G1506775	Component	48T	96T	Storage
G1506775A	Assay Buffer	70 mL	70 mL×2	2-8℃
G1506775B	6-Phosphogluconic Acid	1EA	1EA	2-8℃. Store in the dark.
G1506775C	NADP⁺	1EA	1EA	2-8℃. Store in the dark.

Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

Required Materials Not Provided

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

2. 96-well UV microplate or micro quartz cuvettes, Adjustable pipettes and tips

3. Deionized water

4. Homogenizer (for tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation Steps	Notes & Storage
Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
6-Phosphogluconic Acid Working Regent	Before use, dissolve the vial contents in 2.4 mL Assay Buffer. Mix uniformly.	Keep on ice during use. Store prepared solution at 4°C protected from light for up to 1 week.
NADP⁺ Working Regent	Before use, dissolve the vial contents in 2.4 mL Assay Buffer. Mix uniformly.	Keep on ice during use. Store prepared solution at 4°C protected from light for up to 1 week.

2. Sample Preparation

2.1 Animal Tissue

• Weigh about 0.1 g of tissue.

• Add 1 mL of Assay Buffer and homogenize in an ice bath.

• Centrifuge at 8,000 g, 4°C for 10 minutes.

• Collect the supernatant and keep on ice for assay.

2.2 Plant Tissue

• Weigh about 0.1 g of tissue.

• Add 1 mL of Assay Buffer, mash, and homogenize in an ice bath.

• Centrifuge at 8,000 g, 4°C for 10 minutes.

• Collect the supernatant and keep on ice for assay.

2.3 Cells or Bacteria

• Collect 20 million cells or bacteria into a centrifuge tube.

• Wash cells with cold PBS, centrifuge at 12,000 g, 4°C for 1 min, discard supernatant.

• Add 1 mL of Assay Buffer.

• Sonicate cells or bacteria on ice for 5 minutes (20% power or 200 W, 3s pulse, 7s interval, repeat 30 times).

• Centrifuge at 8,000 g, 4°C for 10 minutes.

• Collect the supernatant and keep on ice for assay.

2.4 Serum (Plasma) and Other Liquid Samples

• Assay directly.

Note: If measuring protein concentration is required, the use of the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

3. Assay Procedure

3.1 Instrument Preparation

• Preheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm.

• Zero the UV spectrophotometer with deionized water.

3.2 Buffer Pre-warming

• Pre-warm the Assay Buffer in a 25°C or 37°C water bath for at least 30 minutes.

3.3 Assay Setup

• Use a 96-well UV plate. Add reagents as follows:

Reagent (μL)	Blank Well	Test Well
Sample	0	20
Deionized Water	20	0
Assay Buffer	140	140
6-Phosphogluconic Acid Working Regent	20	20
NADP⁺ Working Regent	20	20

3.4 Measurement

Mix well after addition.

Measure the absorbance change at 340 nm over 3 minutes.

For the Blank well: Record absorbance at 10 seconds as A₁, and at 190 seconds as A₂. Calculate ΔA_blank = A₂ - A₁. 

For the Test well: Record absorbance at 10 seconds as A₃, and at 190 seconds as A₄. Calculate ΔA_test = A₄ - A₃. 

Note: A pilot experiment with 2-3 variable samples is recommended. 

If ΔA_test is less than 0.002, consider increasing the sample amount. 

If ΔA_test is greater than 0.6, dilute the sample further with Assay Buffer and multiply the result by the dilution factor, or reduce the sample amount used for extraction.

4. Calculation of Results

Note: We provide both the derived formula and the simplified formula for your convenience. They are mathematically equivalent. The bolded simplified formula is recommended for final calculations.

4.1 Calculation using a 96-well UV Plate

(1) Based on Liquid Volume:

Definition of Unit (U): One unit is the amount of enzyme that catalyzes the production of 1 μmol of NADPH per minute per milliliter of liquid sample.

Derived Formula: G6PDH Activity (U/mL) = [(ΔA_test - ΔA_blank) × V_{total reaction} ÷ (ε × d) × 10⁶] ÷ V_sample ÷ T Simplified Formula: G6PDH Activity (U/mL) = 1.0718 × (ΔA_test - ΔA_blank)

(2) Based on Protein Concentration:

Definition of Unit (U): One unit is the amount of enzyme that catalyzes the production of 1 μmol of NADPH per minute per milligram of protein.

Derived Formula: G6PDH Activity (U/mg prot) =[(ΔA_test - ΔA_blank) × V_{total reaction} ÷ (ε × d) × 10⁶] ÷ (C_pr × V_sample) ÷ T 

Simplified Formula: G6PDH Activity (U/mg prot) = 1.0718 × (ΔA_test - ΔA_blank) ÷ C_pr

(3) Based on Sample Fresh Weight:

Definition of Unit (U): One unit is the amount of enzyme that catalyzes the production of 1 μmol of NADPH per minute per gram of tissue (fresh weight).

Derived Formula: G6PDH Activity (U/g) =[(ΔA_test - ΔA_blank) × V_{total reaction} ÷ (ε × d) × 10⁶] ÷ (V_sample ÷ V_{total extract} × W) ÷ T 

Simplified Formula: G6PDH Activity (U/g) = 1.0718 × (ΔA_test - ΔA_blank) ÷ W

(4) Based on Bacterial or Cell Density:

Definition of Unit: One unit of enzyme activity is defined as the amount that generates 1 μmol of NADPH per minute per 10⁴ bacteria or cells.

Derived Formula: G6PDH Activity (μmol/min/10⁴) =[(ΔA_test - ΔA_blank) × V_{total reaction} ÷ (ε × d) × 10⁶] ÷ (2000 × V_sample ÷ V_{total extract}) ÷ T 

Simplified Formula: G6PDH Activity (μmol/min/10⁴) = 0.0005359 × (ΔA_test - ΔA_blank)

Parameter 

Definitions: ΔA_test: A₄ - A₃ (The change in absorbance for the Test well between 190s and 10s) 

ΔA_blank: A₂ - A₁ (The change in absorbance for the Blank well between 190s and 10s) 

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

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

10⁶: Conversion factor (1 mol = 10⁶ μmol) 

V_sample: Volume of sample added to the reaction well, 0.02 mL 

V_{total reaction}: Total volume of the reaction mixture, 0.0002 L 

C_pr: Sample protein concentration, mg/mL 

V_{total extract}: Total volume of Assay Buffer added during sample preparation, 1 mL 

W: Sample fresh weight, g 

T: Reaction time, 3 min 

2,000: Total number of bacteria or cells, in units of 10⁴ (e.g., 20 million cells = 2000 × 10⁴)

4.2 Calculation using a Micro Quartz Cuvette

Use the formulas above but adjust the light path d from 0.5 cm to 1 cm.

Precautions

1. It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

2. This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.

Specifications

Synonyms

Glucose-6-Phosphate Dehydrogenase (G-6-PD) Activity Assay Kit

Specifications & Purity

BioReagent

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Storage

Store at 2-8°C, Protected from light

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive