Hexokinase (HK) Activity Assay Kit (UV Micro Method) - BioReagent, high purity

Cat. No.: H1501279

GRADE & PURITY BioReagent

Descripción general

Introduction
Hexokinase (HK, EC 2.7.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which serves as the intersection point of glycolysis and the pentose phosphate pathway.

Assay Principle
HK catalyzes the synthesis of Glucose-6-Phosphate (G6P) from Glucose. Glucose-6-Phosphate Dehydrogenase (G6PDH) then further catalyzes the dehydrogenation of G6P, generating NADPH. NADPH has a characteristic absorption peak at 340 nm.

Component	48T	96T	Storage
Extraction Buffer	60 mL	60 mL×2	2-8℃
Reagent Ⅰ	14 mL	28 mL	2-8℃
Reagent Ⅱ	1EA	1EA	-20℃. Store in the dark.
Reagent Ⅲ	1EA	1EA	-20℃. Store in the dark.

Note: Please check the quantity of all components before starting the experiment. An additional 10% of each component is provided for standard curve preparation or pilot experiments.

Required Materials and Equipment (Not Provided)

Type	Name	Notes
Instrument	Microplate Reader	Must be capable of measuring absorbance at 340 nm
Consumables	96-well UV Plate	UV-transparent plate
Reagents	Physiological Saline	For sample washing
Other	Homogenizer (for tissue samples), Incubator, Ice box, Refrigerated centrifuge, Adjustable pipettes and tips	Using a multichannel pipette is recommended for high-throughput experiments to improve efficiency

Instructions for Use

1. Reagent Preparation

试剂名称 Reagent Name	Preparation	Notes
Extraction Buffer	Ready-to-use; equilibrate to room temperature (RT) before use.	Store at 4°C
Reagent Ⅰ	Ready-to-use; equilibrate to room temperature (RT) before use.	Store at 4°C
Working Reagent Ⅱ	Prepare immediately before use: • For 48T: Dissolve contents of Reagent Ⅱ in 10.8 mL Reagent Ⅰ • For 96T: Dissolve contents of Reagent Ⅱ in 21.6 mL Reagent Ⅰ	Keep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles.
Working Reagent Ⅲ	Prepare immediately before use: 1. Dissolve Reagent Ⅲ:   • For 48T: in 0.5 mL Reagent Ⅰ   • For 96T: in 1 mL Reagent Ⅰ 2. Dilute the dissolved Reagent Ⅲ 10-fold with Reagent Ⅰ to make the Working Reagent Ⅲ.	Keep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles.

2. Sample Preparation

Note: The use of fresh samples is highly recommended. HK activity decreases significantly upon sample freezing.

2.1 Animal/Plant Tissues
Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.

2.2 Cells, Bacteria, or Fungi
Collect 5×10⁶ cells/bacteria/fungi. Wash with pre-cooled physiological saline and centrifuge at 800 g for 2 min. Discard the supernatant. Add 1 mL of Extraction Buffer and disrupt the cells by sonication on ice (5 min total, 20% power or 200 W, pulse 3s on/7s off, repeat 30 times). Centrifuge the lysate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.

2.3 Serum (or Plasma)
Assay directly.

3. Assay Procedure

3.1 Microplate Reader Preparation: Preheat for at least 30 min. Set the wavelength to 340 nm.
3.2 Working Solution Preparation: Prepare immediately before use. Each well requires 190 µL of Working Solution. It is recommended to prepare enough for 2 extra wells to account for pipetting loss.

For a single well: Mix 180 µL of Working Reagent Ⅱ with 10 µL of Working Reagent Ⅲ.

The Working Solution must be prepared fresh. Incubate it at 37°C for 10 min before the assay.

3.3 Assay Setup: Pipette into wells of the 96-well UV plate as follows:

Reagent	Test Well (µL)
Sample	10
Working Solution	190

3.4 Absorbance Measurement: Immediately after adding the Working Solution, mix thoroughly and measure the absorbance at 340 nm at 10 seconds (A₁) and again after exactly 10 minutes of incubation at 37°C (at 10 min 10 sec, A₂).

4. Result Calculation

The following derived and simplified calculation formulas are provided and are equivalent.

4.1 Data Processing
Calculate ΔA = A₂ - A₁.

4.2 Sample HK Activity Calculation

① Based on Sample Mass (U/g)

Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of sample.

Calculation:
HK (U/g) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × W) ÷ T × n
Simplified Formula: HK (U/g) = 643.09 × ΔA ÷ W × n

② Based on Cell/Bacteria/Fungi Count (U/10⁴)

Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells/bacteria/fungi.

Calculation:
HK (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × 500) ÷ T × n
Simplified Formula: HK (U/10⁴) = 643.09 × ΔA ÷ 500 × n

③ Based on Liquid Volume (U/mL)

Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milliliter of liquid.

Calculation:
HK (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T × n
Simplified Formula: HK (U/mL) = 643.09 × ΔA × n

④ Based on Protein Concentration (U/mg prot)

Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milligram of protein.

Calculation:
HK (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T × n
Simplified Formula: HK (U/mg prot) = 643.09 × ΔA ÷ Cpr × n

Parameter Description:

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

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

• 10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol)

• Vₜₒₜₐₗ: Total reaction volume = 200 μL = 2 × 10⁻⁴ L

• Vₛₐₘₚₗₑ: Volume of supernatant added to the reaction = 10 μL = 1 × 10⁻⁵ L

• Vₛₐₘₚₗₑₜₒₜₐₗ: Volume of Extraction Buffer added = 1 mL

• Cpr: Sample protein concentration (mg/mL)

• W: Sample mass (g)

• T: Reaction time = 10 min

• 500: Cell/Bacteria/Fungi count = 5 × 10⁶, expressed in units of 10⁴

• n: Sample dilution factor

Precautions

1. Before formal testing, it is recommended to perform a pilot experiment using 2-3 samples expected to have significant activity differences.

2. For tissue and cell samples, protein concentration measurement can be used to normalize results between samples.

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

4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear a lab coat, mask, gloves, head cover, and other protective equipment. Perform experiments in a fume hood or biosafety cabinet.

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

Frequently Asked Questions (FAQ)

1. What should I do if the measured ΔA is too high or too low?

If ΔA > 0.5, the sample HK activity is too high. Dilute the supernatant appropriately with Extraction Buffer (include the dilution factor *n* in the calculation) or shorten the reaction time to 5 min.

If ΔA < 0.005, the sample HK activity is too low. Increase the sample volume (keep the Working Solution volume constant and adjust the variable in the formula accordingly) or extend the reaction time (e.g., to 15 or 30 min).

2. Can multiple samples be assayed simultaneously for high-throughput detection?
The initial reaction rate is fast. It is not recommended to run a large number of samples simultaneously. If a multichannel pipette is unavailable, it is best for two individuals to perform the experiment together—one person timing and the other measuring the absorbance—to ensure the accuracy of the results.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Protected from light,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

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