BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Glucokinase (GK, EC 2.7.1.2) is a member of the hexokinase family, primarily found in mature hepatocytes and pancreatic islet cells. Under normal physiological conditions, the main role of GK is to monitor blood glucose levels.
Detection Principle: Glucokinase (GK) phosphorylates glucose to produce glucose-6-phosphate. This product is further coupled with glucose-6-phosphate dehydrogenase and NADP⁺. The increase in NADPH absorbance at 340 nm is measured, allowing for the calculation of the enzyme's activity.
| Component | 100T | Storage |
| Extraction Buffer | 120 mL | 2-8℃ |
| Reagent 1 | 20 mL | 2-8℃ |
| Reagent 2 | 1EA | -20℃ |
| Reagent 3 | 1EA | 2-8℃ |
Reagent 2 (Powder, 1 vial) Preparation:
Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube (can be flicked manually).
Add 1.1 mL of distilled water to dissolve. Use after preparation.
The prepared solution can be stored for the duration of the kit's validity period.
Reagent 3 (Powder, 1 vial) Preparation:
Before opening, ensure the powder is at the bottom (can be flicked manually).
Add 18 mL of Reagent 1 to dissolve. Use after preparation.
The prepared solution can be stored for the duration of the kit's validity period.
User-Prepared Instruments and Materials
Mortar (Homogenizer), Ice box (Ice maker), Benchtop centrifuge, Adjustable micropipettes, Water bath (Oven, Incubator, Metal bath), 96-well plate, Centrifuge tubes, Microplate reader, Distilled water (Deionized water or Ultrapure water are also acceptable).
Experimental Procedure
It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.
1. Sample Extraction
1.1 Tissue Samples
Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, maintain a tissue mass (g) to Extraction Buffer volume (mL) ratio between 1:5 and 1:10.
1.2 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 20% or 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to Extraction Buffer volume (mL) ratio between 500:1 and 1000:1.
1.3 Liquid Samples
Assay directly. If turbid, centrifuge and use the supernatant for assay.
2. Assay Steps
2.1 Preheat the microplate reader for at least 30 minutes. Set the wavelength to 340 nm.
2.2 Pre-warm the prepared Reagent 2 and Reagent 3 at 25°C for 5 minutes to reach room temperature.
2.3 Add reagents sequentially to a 96-well plate:
| Reagent | Test Well (μL) |
| Sample | 20 |
| Reagent 2 | 10 |
| Reagent 3 | 170 |
Mix thoroughly. Read the absorbance at 340 nm at 1 minute (A₁) and again at 21 minutes (A₂, i.e., after 20 minutes of reaction). Calculate ΔA = A₂ - A₁.
Note:
If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer before reading A₂. If the reaction time is changed, the new time (T) must be substituted into the calculation formula. Alternatively, the sample volume can be increased; the new sample volume (V₁) must then be substituted into the calculation formula.
If the increase trend is unstable, read the absorbance every 10 seconds and select a linearly increasing time period for calculation. The corresponding A values for this period should be used to calculate ΔA and substituted into the formula.
3. Calculation of Results
3.1 Based on Sample Protein Concentration
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per mg of tissue protein.
Derived Formula: GK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (V₁ × Cpr) ÷ T
Simplified Formula: GK (nmol/min/mg prot) = 160.77 × ΔA ÷ Cpr
3.2 Based on Sample Fresh Weight
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per gram of tissue.
Derived Formula: GK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ ÷ V) ÷ T
Simplified Formula: GK (nmol/min/g fresh weight) = 160.77 × ΔA ÷ W
3.3 Based on Bacterial or Cell Density
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per 10⁴ bacteria or cells.
Derived Formula: GK (nmol/min/10⁴ cells) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ ÷ V) ÷ T
Simplified Formula: GK (nmol/min/10⁴ cells) = 0.32 × ΔA
3.4 Based on Liquid Volume
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per mL of liquid.
Derived Formula: GK (nmol/min/mL) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ V₁ ÷ T
Simplified Formula: GK (nmol/min/mL) = 160.77 × ΔA
Parameter Definitions:
ε: Molar extinction coefficient of NADPH (6.22 × 10³ L/mol/cm)
d: Light path length for the 96-well plate (0.5 cm)
V: Volume of Extraction Buffer added (1 mL)
V₁: Volume of sample added to the reaction (0.02 mL)
V₂: Total volume of the reaction system (0.2 mL = 2.0 × 10⁻⁴ L)
T: Reaction time (20 minutes)
W: Sample weight (g)
500: Total number of bacteria or cells (5 million)
Cpr: Sample protein concentration (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
Precautions
It is strongly recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.
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