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.
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 decomposition process, catalyzing the conversion of glucose to glucose-6-phosphate. Glucose-6-phosphate is the intersection point of glycolysis and the pentose phosphate pathway.
Detection Principle: HK catalyzes the synthesis of glucose-6-phosphate from glucose. Glucose-6-phosphate dehydrogenase then further catalyzes the dehydrogenation of glucose-6-phosphate, generating NADH. The generated NADH reduces WST-8 to produce an orange-yellow formazan, which has a maximum absorption peak around 450 nm. The HK activity in the sample is calculated by detecting the rate of increase in light absorption at 450 nm.
Detection Range: 31.25 - 2000 µM
Sensitivity: 31.25 µM
Applicable Samples: Animal/plant tissues, cells, cell supernatant, bacteria, serum (plasma).
| H1501177 | Component | 48T | 96T | Storage |
| H1501177A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| H1501177B | Assay Buffer | 16 mL | 32 mL | 2-8℃ |
| H1501177C | Substrate Mix | 1EA | 1EA | 2-8℃. Store in the dark. |
| H1501177D | Enzyme Mix | 45 µL | 90 µL | -20℃. Store in the dark. |
| H1501177E | WST-8 | 400 μL | 800 μL | -20℃. Store in the dark. |
| H1501177F | Enhancer | 80 µL | 160 µL | -20℃. Store in the dark. |
| H1501177G | NADH Standard | 1EA | 1EA×2 | -20℃. Store in the dark. |
Please check the quantity of each component before the experiment.
An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.
User-Provided Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 450 nm. |
| Consumables | 96-well Microplate | Standard transparent plate. |
| Reagents | PBS / Deionized Water | For washing samples / Preparing standards. |
| Others | Homogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tips | Using a multichannel pipette for large-scale detection can improve efficiency. |
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 | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Substrate Mix | Prepare before use: For 48T: Dissolve in 15.2 mL Assay Buffer. For 96T: Dissolve in 30.4 mL Assay Buffer. | Unused dissolved Substrate Mix can be stored at 4°C for one week. For longer storage, aliquot and store at -20°C protected from light for 1 month. Avoid repeated freeze-thaw cycles. |
| Enzyme Mix | Ready-to-use; equilibrate to room temperature before use. | Protect from light during the experiment; store at -20°C protected from light. |
| WST-8 | Ready-to-use; equilibrate to room temperature before use. | Protect from light during the experiment; store at -20°C protected from light. |
| Enhancer | Ready-to-use; equilibrate to room temperature before use. | Protect from light during the experiment; store at -20°C protected from light. |
| NADH Standard | Prepare before use: Dissolve in 1 mL deionized water. Mix well. | Concentration is 2000 µM. Unused dissolved standard can be stored at 4°C for one week. For longer storage, aliquot and store at -20°C protected from light for 1 month. Avoid repeated freeze-thaw cycles. |
2. Standard Preparation
Dilute the 2000 µM standard with deionized water to prepare standard solutions of 1000, 500, 250, 125, 62.5, and 31.25 µM.
A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
| Standard Working Solution | Standard (μL) | Deionized Water (μL) | Concentration (µM) |
| Std.1 | 200µL of 2000μM | 0 | 2000 |
| Std.2 | 100µL of 2000μM | 100 | 1000 |
| Std.3 | 100µL of 1000μM | 100 | 500 |
| Std.4 | 100µL of 500μM | 100 | 250 |
| Std.5 | 100µL of 250μM | 100 | 125 |
| Std.6 | 100µL of 125μM | 100 | 62.5 |
| Std.7 | 100µL of 62.5μM | 100 | 31.25 |
| Blank | 0 | 100 | 0 |
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.
3.1 Animal Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
3.2 Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and grind. Disrupt by ultrasonic homogenization on ice (power 20%, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
3.3 Cells/Bacteria: Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with pre-cooled PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20%, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
3.4 Cell Supernatant or Serum (Plasma): Detect directly.
4. Assay Steps
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 450 nm.
4.2 Assay Working Reagent Preparation: For each test well, prepare 190 µL of Assay Working Reagent by pipetting 183 µL Substrate Mix, 1 µL Enzyme Mix, 5 µL WST-8, and 1 µL Enhancer. Prepare freshly before use.
4.3 Control Working Reagent Preparation: For each control well, prepare 190 µL of Control Working Reagent by pipetting 184 µL Substrate Mix, 5 µL WST-8, and 1 µL Enhancer. Prepare freshly before use.
4.4 Assay System Setup:
Blank Well: Add 10 µL deionized water and 190 µL Assay Working Reagent.
Standard Well: Add 10 µL of various standard solutions and 190 µL Assay Working Reagent.
Test Well: Add 10 µL of sample and 190 µL Assay Working Reagent.
Control Well: Add 10 µL of sample and 190 µL Control Working Reagent.
Note: Set up one Control Well for each sample to eliminate interference from the sample's own color.
4.5 Mix the reaction system thoroughly and incubate at room temperature for 5 minutes.
4.6 Absorbance Measurement: Read the absorbance at 450 nm, recorded as A blank , A standard , A test , and A control .
5. Result Calculation The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.
5.1 Data Processing Calculate ΔA standard = A standard - A blank , ΔA test = A test - A control .
5.2 Standard Curve Plotting
Plot the standard curve with standard concentration as the y-axis and ΔA standard as the x-axis. Substitute ΔA test into the equation to obtain the y value (1 µM = 1 nmol/mL), which is the NADH content.
5.3 Sample HK Activity Calculation
(1) Based on sample mass:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADH per minute per gram of tissue.
Formula: HK (U/g fresh weight) = y × V standard ÷ V sample × V extract ÷ W ÷ T = 0.2 × y ÷ W
(2) Based on cell or bacterial count:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADH per minute per 10⁴ cells or bacteria.
Formula: HK (U/10⁴) = y × V standard ÷ V sample × V extract ÷ 500 ÷ T = 0.0004 × y
(3) Based on liquid volume:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADH per minute per mL of liquid.
Formula: HK (U/mL) = y × V standard ÷ V sample ÷ T = 0.2 × y
(4) Based on protein concentration:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADH per minute per mg of protein.
Formula: HK (U/mg prot) = y × V standard ÷ V sample ÷ Cpr ÷ T = 0.2 × y ÷ Cpr
Parameter Description:
1 µM = 1 nmol/mL;
V standard : Volume of standard added, 0.01 mL;
V sample : Volume of sample added, 0.01 mL;
V extract : Volume of Extraction Buffer added, 1 mL;
W: Sample mass, g;
T: Reaction time, 5 min;
Cpr: Sample protein concentration, mg/mL;
500: Cell or bacterial count (5 × 10⁶), converted to units of 10⁴.
6. Result Presentation
Typical Standard Curve: y = 1304.2x + 5.7163, R² = 0.9999
纵坐标的标准品浓度改成“Concentration of Standard”
Example: 0.1 g of mouse muscle tissue was processed and assayed according to the procedure using a 96-well plate.
Measured: ΔA test = A test - A control = 0.562 - 0.065 = 0.497.
Substituting into the standard curve equation gives y = 653.9037.
Calculated based on sample mass:
HK (U/g fresh weight) = 0.2 × y ÷ W = 1307.81 U/g.
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 tissue and cell samples, results can be normalized by measuring the protein concentration. Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
4. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.
5. 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.
6. This product is for scientific research use only. Not intended for clinical diagnosis.
Frequently Asked Questions
Q: What should I do if the sample ΔA test is too high or too low?
A: If the sample ΔA test is > 1.5, the HK activity in the sample is too high. Dilute the sample appropriately with Extraction Buffer and re-assay. If the sample ΔA test is < 0.001, increase the sample amount and re-assay.
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