BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
己糖激酶（HK，EC2.7.1.1）广泛存在于动物、植物、微生物和培养细胞中，是葡萄糖分解过程中的第一个关键酶，催化葡萄糖转化为6-磷酸葡萄糖，6-磷酸葡萄糖是糖酵解和磷酸戊糖途径的交叉点。
检测原理：HK催化葡萄糖合成6-磷酸葡萄糖，6-磷酸葡萄糖脱氢酶进一步催化6-磷酸葡萄糖 脱氢生成NADH，生成的NADH将WST-8还原生成橙黄色的formazan（甲臜），在450nm左右检测有最大吸收峰。通过检测450nm处光吸收增加速率即可计算出样本中HK活性。
检测范围：31.25-2000 μM
灵敏度：31.25 μM
适用样本：动植物组织、细胞、细胞上清、细菌、血清（浆）
| 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. |
请在实验前检查各组分的量。
各组分在规格基础上额外提供10%的量用于进行标准曲线制作或预实验。
自备仪器和试剂
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测450nm处的吸光度 |
| 耗材 | 96孔酶标板 | 普通透明板 |
| 试剂 | PBS/去离子水 | 清洗样本用/配制标准品用 |
| 其他 | 匀浆器（组织样本）、恒温箱、冰盒、低温离心机、可调节式移液枪及枪头 | 检测量较大时合理使用多通道移液器有助于提高实验效率 |
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Assay Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Substrate Mix | 临用前配制； 48T加入15.2mL Assay Buffer溶解， 96T加入30.4mL Assay Buffer 溶解 | 未用完的已溶解的Substrate Mix可4℃保存一周。 若需更长时间保存，请分装后-20℃避光保存1个月，避免反复冻融。 |
| Enzyme Mix | 即用型；使用前平衡到室温 | 实验过程中避光放置； -20℃避光保存 |
| WST-8 | 即用型；使用前平衡到室温 | 实验过程中避光放置； -20℃避光保存 |
| Enhancer | 即用型；使用前平衡到室温 | 实验过程中避光放置； -20℃避光保存 |
| NADH Standard | 临用前配制； 用1mL去离子水充分溶解 | 浓度为2000μM； 未用完的已溶解的NADH Standard可4℃保存一周。 若需更长时间保存，请分装后-20℃避光保存1个月，避免反复冻融。 |
2、标准品配制
使用去离子水将浓度为2000μM的标准品稀释为1000μM、500μM、250μM、125μM、62.5μM、31.25μM的标准溶液。每次实验都要做一次标准品检测，制作标曲；稀释后的标准品溶液不稳定，必须在4小时内使用。
| 标准品工作液 | 标准品 (μL) | 去离子水 (μL) | 浓度 (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 |
| 空白 | 0 | 100 | 0 |
3、样本制备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存1个月。对于脂肪含量较高的动物组织，离心后移除上层脂肪，再取上清液。
3.1 动物组织样本：
称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，8000g，4℃离心10min，取上清液置冰上待测。
3.2 植物组织样本：
称取约0.1g样本，加入1mL Extraction Buffer捣碎，冰浴超声破碎5min（功率20%，超声3s，间隔7s，重复30次），8000g，4℃离心10min，取上清液置冰上待测。
3.3 细胞（菌）：
收集5×10⁶个细胞或细菌到离心管内，用预冷的PBS清洗，离心后弃上清。加入1mL Extraction Buffer，冰浴超声破碎5min（功率20%，超声3s，间隔7s，重复30次），然后8000g，4℃离心10min，取上清液置冰上待测。
3.4 细胞上清或血清（浆）：
直接检测。
4、实验步骤
4.1 酶标仪准备：预热30min以上，调节波长到450nm。
4.2 测定工作液的配制：
测定孔每孔配制190µL测定工作液：吸取183µL Substrate Mix，1µL Enzyme Mix，5µL WST-8，1µL Enhancer。测定工作液需现配现用。
4.3 对照工作液的配制：
对照孔每孔配制190µL对照工作液：吸取184µL Substrate Mix，5µL WST-8，1µL Enhancer。对照工作液需现配现用。
4.4 检测体系配制：
空白孔在96孔板中依次加入10μL去离子水和190μL测定工作液；
标准孔在96孔板中依次加入10μL不同浓度的标准品和190μL测定工作液；
测定孔在96孔板中依次加入10μL样本和190μL测定工作液；
对照孔在96孔板中依次加入10μL样本和190μL对照工作液。
注意：每个样本设置一个对照孔，消除样本本身颜色的干扰。
4.5 反应体系充分混匀后，室温孵育5min。
4.6 吸光度检测：在450nm处读取吸光度值，分别标记为A 空白 、A 标准 、A 测定 和A 对照 。
5、结果计算 以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
5.1 数据处理 计算ΔA 标准 =A 标准 -A 空白 ，ΔA 测定 =A 测定 -A 对照 。
5.2 标准曲线的绘制 以标准液浓度为y轴，ΔA 标准 为x轴，绘制标准曲线。将ΔA 测定 带入方程得到y值 (1µM=1nmol/mL) 即NADH含量。
5.3 样本HK活性计算
(1) 按样本质量计算
单位的定义：每g组织每分钟生成1nmol的NADH定义为一个酶活力单位。
HK (U/g 鲜重)=y×V 标准 ÷V 样 ×V 提取 ÷W÷T=0.2×y÷W
(2) 按细胞或细菌数量计算
单位的定义：每10⁴个细胞或细菌每分钟生成1nmol的NADH定义为一个酶活力单位。
HK (U/10⁴)=y×V 标准 ÷V 样 ×V 提取 ÷500÷T=0.0004×y
(3) 按液体体积计算
单位的定义：每毫升液体每分钟生成1nmol的NADH定义为一个酶活力单位。
HK (U/mL)=y×V 标准 ÷V 样 ÷T=0.2×y
(4) 按蛋白浓度数量计算
单位的定义：每mg蛋白每分钟生成1nmol的NADH定义为一个酶活力单位。
HK (U/mg prot)=y×V 标准 ÷V 样 ÷Cpr÷T=0.2×y÷Cpr
参数说明
1 μM=1 nmol/mL；
V 标准 ：加入标准品体积，0.01mL；
V 样 ：加入样本体积，0.01mL；
V 提取 ：加入Extraction Buffer 体积，1mL；
W：样本质量，g；
T：反应时间，5min；
Cpr：样本蛋白质浓度，mg/mL；
500：细胞或细菌数量，5×10⁶，以10⁴为单位。
6、结果展示
典型标准曲线：y=1304.2x+5.7163，R²=0.9999
示例：取0.1g小鼠肌肉组织，按照测定步骤操作，用96孔板进行检测。测得：ΔA 测定 =A 测定 -A 对照 =0.562-0.065=0.497。将ΔA 测定 代入标准曲线方程，得到y=653.9037。按样本质量计算得：HK (U/g鲜重)=y×V 标准 ÷V 样 ×V 提取 ÷W÷T=0.2×y÷W=1307.81 U/g。
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
3、对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
4、建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线，可参考结果展示部分的典型标准曲线公式进行结果计算。
5、生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护，穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
6、本产品仅供科学研究使用，不适用于临床诊断。
常见问题及解答
Q：检测得到的样本ΔA 测定 过高或者过低怎么办？
A：如果样本的ΔA 测定 ＞1.5，则说明样本HK活性太高，需用Extraction Buffer适当稀释，再重新进行测定。如果样本的ΔA 测定 ＜0.001，可提高样本量，再重新进行测定。
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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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1028976 |
分析证书 | H1501177 |
危险品化学品经营许可证（带存储）