计算溶液所需的质量、体积或浓度。
葡萄糖氧化酶活性检测试剂盒 (铜皂, 微量法)
有货
为什么选择此级别
BioReagent,标曲法 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
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。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
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。
概述
葡萄糖氧化酶（Glucose Oxidase，GOD）(EC 1.1.3.4)是一种二聚酶，催化β-D-葡萄糖氧化成过氧化氢和水解成葡萄糖酸的 D-葡萄糖-1,5-内酯。这种酶是由某些种类的昆虫、真菌和细菌产生的。葡萄糖氧化酶广泛用于测定体液中的葡萄糖，以及从饮料、食品和其他农产品中去除残留的葡萄糖和氧气。此外，葡萄糖氧化酶通常用于生物传感器来检测葡萄糖。
本试剂盒检测原理是 GOD 催化葡萄糖产生H₂O₂ ，H₂O₂在酸性条件下与指示剂反应产生一种紫色产物，580nm 处的吸光度与生成H₂O₂的浓度成正比，也就是与 GOD 活性成正比。
组分表
| 组分货号 | 组分名称 | 物理外观 | 96 T | 480T | 储存 |
| G1527314A | 分析缓冲液 (20×) | 液体 | 10mL | 50mL | 2-8℃. |
| G1527314B | 葡萄糖 (0.2 M) | 液体 | 7mL | 35mL | -20℃ |
| G1527314C | H₂O₂ 标准品 (0.88 M) | 液体 | 100μL | 100μL | -20℃.避光 |
| G1527314D | 显色底物 | 液体 | 5.6mL | 28mL | -20℃.避光 |
| G1527314E | 葡萄糖氧化酶 (对照) | 液体 | 100μL | 100μL | -20℃.避光 |
*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。
自备仪器与试剂
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测 580 nm 处的吸光度 |
| 耗材 | 96 孔酶标板 | 普通透明板 |
| 试剂 | 去离子水/PBS | 试剂配置用/样本清洗用 |
| 其他 | 匀浆器（组织样本） 、低温离心机、恒温箱、制冰机、 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 |
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. 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。
3. 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。
4. 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。
5. 本产品仅供科学研究使用，不适用于临床诊断
试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
|---|---|---|
| 1×分析缓冲液 | 使用前平衡到室温；用去离子水按 1:20 稀释 分析缓冲液 (20×) 得到 1×分析缓冲液 | 1×分析缓冲液 可用于稀释 H₂O₂ 标准品和样本；未用完的试剂可在 4℃ 保存至少两个月 |
| 葡萄糖 (0.2 M) | 即用型；使用前平衡到室温 | 建议分装后 -20℃ 保存 |
| H₂O₂ 标准品 (0.88 M) | 即用型 | 整个实验过程中 避光 放置；建议分装后 -20℃ 避光 保存 |
| 显色底物 | 即用型 | 整个实验过程中 避光 放置；建议分装后 -20℃ 避光 保存；有毒且有刺激性气味，建议在通风橱中进行实验 |
工作型葡萄糖氧化酶 (对照) | 提供的葡萄糖氧化酶来自黑曲霉，作为阳性对照。 ① 取2 μL葡萄糖氧化酶 (对照)，用 998 μL 1×分析缓冲液 进行第一步稀释，混匀； ② 再取1 μL第一步稀释的溶液，用399 μL 1×分析缓冲液 稀释，得到 工作型葡萄糖氧化酶 (对照) | 整个实验过程中 冰上避光 放置；未稀释的试剂建议分装后 -20℃ 避光 保存 |
标准品配制
配制 10 mM H₂O₂ 标准品：10 μL 0.88 M 的H₂O₂ 标准品 用 870 μL 的 1×分析缓冲液 稀释。配制 40 μM H₂O₂ 标准品：4 μL 10 mM 的H₂O₂ 标准品 用 996 μL 的 1×分析缓冲液 稀释。使用 40 μM H₂O₂ 标准品，按照下表所示，进一步稀释标准品：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。
| 序号 | 40 μM 标准品体积 (μL) | 1×分析缓冲液 (μL) | 浓度 (μM) |
|---|---|---|---|
| 1 | 200 | 0 | 40 |
| 2 | 150 | 50 | 30 |
| 3 | 100 | 100 | 20 |
| 4 | 50 | 150 | 10 |
| 5 | 25 | 175 | 5 |
| 6 | 10 | 190 | 2 |
| 7 | 5 | 195 | 1 |
| 空白 | 0 | 200 | 0 |
样本制备
注意：样本以新鲜为宜，若不能立刻检测，应储存在-80℃保存 1 个月。
1.动物组织：用冷的 PBS 清洗组织，尽可能去除血液。吸干组织上的水分，称取约 0.1 g 样本，加入预冷的1×分析缓冲液 匀浆。4℃，12000 g 离心 5 min，取上清液置于冰上做进一步分析。
2. 细胞或细菌：收集 5×106 个细胞，用预冷的 PBS 清洗细胞，800 g 离心 2 min，弃上清。用 1 mL 预冷的 1×AssayBuffer 冰浴超声波破碎细胞 5 min（功率 20%或 200 W，超声 3 s，间隔 7 s，重复 30 次）。4℃，12000 g离心 5 min，取上清液置于冰上做进一步分析。
3. 血清（浆）等液体样本：可直接检测。为确保检测值在标准值范围内，建议将样本用 1×分析缓冲液 稀释成不同的浓度后，再进行检测。
实验步骤
1. 酶标仪准备：预热 30 min 以上，调节波长到 580 nm。
2. 检测体系配制：在 1.5 mL EP 管中按照如下式方操作：
| 试剂 | 空白管 (μL) | 标准管 (μL) | 测定管 (μL) | 对照管 (μL) |
|---|---|---|---|---|
| 1×分析缓冲液 | 50 | 0 | 0 | 0 |
| 标准品 | 0 | 50 | 0 | 0 |
| 样本 | 0 | 0 | 50 | 0 |
工作型葡萄糖氧化酶 (对照) | 0 | 0 | 0 | 50 |
| 葡萄糖 (0.2 M) | 50 | 50 | 50 | 50 |
3.吸光度检测：混匀。37℃孵育 20 min，吸取 60 μL 上述混合液到 96 孔板，每孔迅速加入 40 μL 显色底物（建议使用多通道移液器）。轻敲板，充分混合后，37℃避光孵育 10 min，读取 580 nm 处的吸光值 A，分别记为 A空白、A 对照、A标准和 A测定。
结果计算
1.数据处理
计算 ΔA标准 = A标准 − A空白，ΔA测定 = A测定 − A空白t;，ΔA测定= ΔA测定 −A空白
2.标准曲线绘制
以标准品浓度为 y 轴，ΔA标准 为 x 轴，绘制标准曲线。将 ΔA测定 代入方程得到 y 值（μM）。（1 μM = 1 nmol/mL）
3.样本 GOD 活性计算
① 按样本蛋白浓度计算
活性单位定义：每 mg 组织蛋白每分钟生成 1 nmol 的 H₂O₂ 定义为一个酶活力单位。
GOD(U/mg prot)=y÷Cpr÷T=0.05×y÷Cpr
② 按样本鲜重计算
活性单位定义：每 g 组织每分钟生成 1 nmol 的 H₂O₂ 定义为一个酶活力单位。
GOD(U/g)=y÷W÷T=0.05×y÷W
③ 按细胞或细菌数量计算
活性单位定义：每 1 万个细菌或细胞每分钟生成 1 nmol 的 H₂O₂ 定义为一个酶活力单位。
GOD (U/10⁴)=y÷N÷T=0.05×y÷N
④ 按样本体积计算
活性单位定义：每 mL 液体样本每分钟生成 1 nmol 的 H₂O₂ 定义为一个酶活力单位。
GOD(U/mL)=y÷V 样 ÷T=y
| 符号 | 含义 | 单位/取值 |
|---|---|---|
| W | 样品质量 | g |
| T | 反应时间 | 20 min |
| N | 细胞数量（以 10⁴ 为单位） | 例如 5×10⁶ 个细胞，则 N = 500 |
| V 样 | 加入样本体积 | 0.05 mL |
| Cpr | 样本蛋白质浓度 | mg/mL |
Glucose Oxidase (GOD) (EC 1.1.3.4) is a dimeric enzyme that catalyzes the oxidation of β‑D‑glucose to D‑glucose‑1,5‑lactone, which then hydrolyzes to gluconic acid, with the concomitant production of hydrogen peroxide. This enzyme is produced by certain species of insects, fungi, and bacteria. Glucose oxidase is widely used for the determination of glucose in body fluids, as well as for the removal of residual glucose and oxygen from beverages, food products, and other agricultural commodities. In addition, glucose oxidase is commonly employed in biosensors for glucose detection.
Assay Principle of This Kit:GOD catalyzes the oxidation of glucose to generate H₂O₂. In an acidic environment, the generated H₂O₂ reacts with a chromogenic indicator to produce a purple colored product. The absorbance at 580 nm is proportional to the concentration of H₂O₂ produced, which is in turn proportional to the GOD activity in the sample.
| omponent No. | Component Name | Appearance | 96T | 480T | Storage |
| G1527314A | Assay Buffer (20×) | Liquid | 10mL | 50mL | 2-8℃. |
| G1527314B | Glucose (0.2 M) | Liquid | 7mL | 35mL | -20℃ |
| G1527314C | H₂O₂ Standard (0.88 M) | Liquid | 100μL | 100μL | -20℃.Store ai dark. |
| G1527314D | Chromogen | Liquid | 5.6mL | 28mL | -20℃.Store ai dark. |
| G1527314E | Glucose Oxidase (Control) | Liquid | 100μL | 100μL | -20℃.Store ai dark. |
note:
Please check the amount 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.
Reagents, consumables and Equipments not provided
| Type | Resource Name | Remarks |
|---|---|---|
| Instruments | Microplate reader | Capable of measuring absorbance at 580 nm |
| Consumables | 96‑well microplate | Standard transparent plate |
| Reagents | Deionized water / PBS | For reagent preparation / sample washing |
| Others | Homogenizer (for tissue samples), refrigerated centrifuge, incubator, ice maker, adjustable pipettes and tips | Using a multichannel pipette is helpful for improving efficiency when handling a large number of samples |
Precautions
1.Before formal testing, it is recommended to perform preliminary experiments with 2‑3 samples that are expected to show obvious differences.
2.For tissue and cell samples, results can be normalized by measuring protein concentration.
3.It is recommended that users establish their own standard curve to improve accuracy. If not, the typical standard curve formula provided in the results section can be used for calculation.
4.Biochemical assay reagents generally have irritancy and biological toxicity. For your safety and health, please take proper biosafety precautions throughout the experiment, wear lab coats, masks, gloves, head covers and other protective equipment, and perform experiments in a fume hood or biosafety cabinet.
5.This product is for research use only, not for clinical diagnosis.
Reagent Preparation
| Reagent Name | Preparation | Notes |
|---|---|---|
| 1×Assay Buffer | Equilibrate to room temperature before use; dilute Assay Buffer (20×) 1:20 with deionized water to obtain 1×Assay Buffer | 1×Assay Buffer can be used to dilute H₂O₂ standard and samples; unused reagent can be stored at 4°C for at least two months |
| Glucose (0.2 M) | Ready‑to‑use; equilibrate to room temperature before use | Recommended to aliquot and store at ‑20°C |
| H₂O₂ Standard (0.88 M) | Ready‑to‑use | Keep away from light throughout the experiment; recommended to aliquot and store at ‑20°C in the dark |
| Chromogen | Ready‑to‑use | Keep away from light throughout the experiment; recommended to aliquot and store at ‑20°C in the dark; toxic and has a pungent odor, it is recommended to perform experiments in a fume hood |
| Working Glucose Oxidase (Control) | The provided Glucose Oxidase (Control) is from Aspergillus niger and serves as a positive control. ① First, dilute 2 μL of Glucose Oxidase (Control) with 998 μL of 1×Assay Buffer and mix. ② Then take 1 μL of the first dilution and dilute with 399 μL of 1×Assay Buffer to obtain Working Glucose Oxidase (Control) | Keep on ice and away from light throughout the experiment; undiluted reagent is recommended to be aliquoted and stored at ‑20°C in the dark |
Standard Preparation
Prepare 10 mM H₂O₂ Standard: dilute 10 μL of 0.88 M H₂O₂ Standard with 870 μL of 1×Assay Buffer.
Prepare 40 μM H₂O₂ Standard: dilute 4 μL of 10 mM H₂O₂ Standard with 996 μL of 1×Assay Buffer.
Using the 40 μM H₂O₂ Standard, further dilute the standards according to the table below. A standard curve must be prepared in each experiment; the diluted standard solutions are unstable and must be used within 4 hours.
| No. | Volume of 40 μM Standard (μL) | 1×Assay Buffer (μL) | Concentration (μM) |
|---|---|---|---|
| 1 | 200 | 0 | 40 |
| 2 | 150 | 50 | 30 |
| 3 | 100 | 100 | 20 |
| 4 | 50 | 150 | 10 |
| 5 | 25 | 175 | 5 |
| 6 | 10 | 190 | 2 |
| 7 | 5 | 195 | 1 |
| Blank | 0 | 200 | 0 |
Sample Preparation
Note: Fresh samples are preferred. If immediate analysis is not possible, store at ‑80°C for up to 1 month.
1.Animal tissues: Wash the tissue with cold PBS to remove blood as much as possible. Blot dry the tissue, weigh about 0.1 g of sample, and homogenize in pre‑cooled 1×Assay Buffer. Centrifuge at 12,000 g for 5 min at 4°C, and take the supernatant on ice for further analysis.
2.Cells or bacteria: Collect 5×10⁶ cells, wash with pre‑cooled PBS, centrifuge at 800 g for 2 min, and discard the supernatant. Resuspend in 1 mL of pre‑cooled 1×Assay Buffer and disrupt cells by ultrasonic treatment on ice for 5 min (power 20% or 200 W, sonicate 3 s, interval 7 s, repeat 30 times). Centrifuge at 12,000 g for 5 min at 4°C, and take the supernatant on ice for further analysis.
3.Liquid samples such as serum/plasma: Can be assayed directly. To ensure that the measured values fall within the standard range, it is recommended to dilute the sample with 1×Assay Buffer to different concentrations before testing.
Assay Procedure
1.Microplate reader preparation: Preheat for at least 30 min and set the wavelength to 580 nm.
2. Reaction system setup: In 1.5 mL EP tubes, prepare as follows:
| Reagent | Blank tube (μL) | Standard tube (μL) | Sample tube (μL) | Control tube (μL) |
|---|---|---|---|---|
| 1×Assay Buffer | 50 | 0 | 0 | 0 |
| Standard | 0 | 50 | 0 | 0 |
| Sample | 0 | 0 | 50 | 0 |
| Working Glucose Oxidase (Control) | 0 | 0 | 0 | 50 |
| Glucose (0.2 M) | 50 | 50 | 50 | 50 |
3. Absorbance measurement: Mix well. Incubate at 37°C for 20 min. Transfer 60 μL of the above mixture to a 96‑well plate, and quickly add 40 μL of Chromogen to each well (a multichannel pipette is recommended). Tap the plate gently to mix thoroughly, then incubate at 37°C in the dark for 10 min. Read the absorbance at 580 nm, recording as Ablank, Acontrol,Astandard, and Asample.
Calculation of Results
1. Data processing
CalculateΔAstandard = Astandard − Ablank, and ΔAsample = Asample − Ablank.
2. Standard curve plotting
Plot the standard curve with standard concentration on the y‑axis and ΔAstandard on the x‑axis. Substitute ΔA<sub>sample</sub> into the equation to obtain the y‑value (μM). (1 μM = 1 nmol/mL)
3. Calculation of GOD activity in samples
① Based on sample protein concentration
Definition of activity unit: One unit (U) is defined as the amount of enzyme that generates 1 nmol of H₂O₂ per minute per mg of tissue protein.
GOD (U/mg prot) = y ÷ Cpr ÷ T = 0.05 × y ÷ Cpr
② Based on sample fresh weight
Definition: One unit is defined as the amount that generates 1 nmol of H₂O₂ per minute per gram of tissue.
GOD (U/g) = y ÷ W ÷ T = 0.05 × y ÷ W
③ Based on cell or bacterial number
Definition: One unit is defined as the amount that generates 1 nmol of H₂O₂ per minute per 10⁴ cells or bacteria.
GOD (U/10⁴) = y ÷ N ÷ T = 0.05 × y ÷ N
④ Based on sample volume
Definition: One unit is defined as the amount that generates 1 nmol of H₂O₂ per minute per mL of liquid sample.
GOD (U/mL) = y ÷ Vsample ÷ T = 0.05 × y ÷ Vsample
| Symbol | Meaning | Unit / Value |
|---|---|---|
| W | Sample weight | g |
| T | Reaction time | 20 min |
| N | Cell number (in 10⁴ units) | e.g., for 5×10⁶ cells, N = 500 |
| Vsample | Volume of sample added | 0.05 mL |
| Cpr | Sample protein concentration | mg/mL |
规格
规格或纯度
BioReagent, 标曲法
英文名称
Glucose Oxidase Activity Assay Kit (Copper Soap, Micro Method)
应用
酶活性检测
检测范围
1-40 μM
检测方法
分光光度法
检测仪器
酶标仪
检测波长
580nm
敏感性
Light Sensitive
储存与运输
储存条件
2-8°C储存,避光,-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期12个月。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 96T | 480T | 储存 |
| G1527314A | 分析缓冲液 (20×) | 液体 | 10mL | 50mL | 2-8℃. |
| G1527314B | 葡萄糖 (0.2 M) | 液体 | 7mL | 35mL | -20℃ |
| G1527314C | H₂O₂ 标准品 (0.88 M) | 液体 | 100μL | 100μL | -20℃.避光 |
| G1527314D | 显色底物 | 液体 | 5.6mL | 28mL | -20℃.避光 |
| G1527314E | 葡萄糖氧化酶 (对照) | 液体 | 100μL | 100μL | -20℃.避光 |
注：整套试剂盒未拆封于−20 ℃避光保存，自生产之日起有效期 12 个月。拆封后各组分稳定性不同：为避免反复冻融，组分 G1527314A、G1527314B 可转至 2‑8 ℃存放，有效期仍为 12 个月；组分 G1527314C、G1527314D、G1527314E 需继续冷冻保存。
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