Name: β-淀粉酶 (β-AL) 活性检测试剂盒（DNS, 微量法）
SKU: A1508361
Availability: InStock

概述规格储存与运输技术文档高级数据质检证书（CoA,COO,BSE/TSE 和分析图谱) 技术文档和文章溶液计算器

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为什么选择此级别

BioReagent 级，适用于对基线干扰要求严格的色谱和分析工作流程。

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储存与运输

2-8°C储存,避光。低温运输。请查阅批次 COA 获取详细规格。

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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。

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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

淀粉酶负责水解淀粉，主要包括α-淀粉酶（α-AL）和β-淀粉酶（β-AL，EC 3.2.1.2）。β-淀粉酶从淀粉的非还原端切开α-1,4糖苷键，生成葡萄糖、麦芽糖、麦芽三糖、糊精等还原糖。

检测原理：淀粉酶催化淀粉水解生成还原糖，还原糖还原3,5-二硝基水杨酸生成棕红色物质，在540nm有吸收峰；通过测定540nm吸光度增加速率，计算淀粉酶活性。α-淀粉酶耐热，但是β-淀粉酶在70℃下15 min可钝化。因此粗酶液经过70℃钝化15min，就只有α-淀粉酶能够催化淀粉水解。用不钝化的粗酶液测出总淀粉酶活性，减去α-淀粉酶就可得到β-淀粉酶活性。

检测范围：0.0156-1 mg/mL

灵敏度：0.0078 mg/mL

适用样本：血清（浆）、唾液、动植物组织

注意：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。

自备仪器和试剂

酶标仪或可见分光光度计（能测540nm处的吸光度）
96孔板或微量玻璃比色皿、可调节式移液枪及枪头
低温离心机、水浴锅、制冰机、恒温箱
去离子水
匀浆器（如果是组织样本）

操作步骤

1、试剂准备

试剂名称	试剂准备	注意事项
DNS Reagent	即用型，使用前平衡到室温	4℃避光保存
Substrate	临用前96T加入42mL去离子水，48T加入21mL去离子水，上下颠倒摇晃几次，95℃加热至溶解。	溶解后的Substrate 4℃保存一周，若有沉淀析出，可95℃加热溶解后使用。
Standard	含10mg无水葡萄糖，临用前加入1mL去离子水溶解得10mg/mL标准品。	溶解后的标准品可4℃保存2周。

2、标准曲线设置

按下表所示，用去离子水将10mg/mL标准品稀释为1、0.5、0.25、0.125、0.0625、0.0313、0.0156 mg/mL的标准溶液。

序号	标准品体积	去离子水体积（µL）	标准品浓度（mg/mL）
Std.1	40µL 10mg/mL	360	1
Std.2	200µL of Std.1 (1mg/mL)	200	0.5
Std.3	200µL of Std.2 (0.5mg/mL)	200	0.25
Std.4	200µL of Std.3 (0.25mg/mL)	200	0.125
Std.5	200µL of Std.4 (0.125mg/mL)	200	0.0625
Std.6	200µL of Std.5 (0.0625mg/mL)	200	0.0313
Std.7	200µL of Std.6 (0.0313mg/mL)	200	0.0156

注意：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。

3、样本制备

注意：推荐使用新鲜样本。

3.1 动物组织

称取约0.1g样本，加入1mL去离子水匀浆，将匀浆倒入离心管中，在室温下放置15min，每隔5 min振荡1次，使其充分提取。6,000g，室温离心10min，吸取上清液并且加去离子水定容至10mL，摇匀，即为淀粉酶原液。吸取上述淀粉酶原液1mL，加入4mL去离子水，摇匀，即为淀粉酶稀释液，用于(α＋β)淀粉酶总活力的测定。

3.2 植物组织

称取约0.1g样本，加入1mL去离子水捣碎，超声波破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），在室温下放置15min，每隔5min振荡1次，使其充分提取。6,000g，室温离心10min，吸取上清液并且加去离子水定容至10mL，摇匀，即为淀粉酶原液。吸取上述淀粉酶原液1mL，加入4mL去离子水，摇匀，即为淀粉酶稀释液，用于(α＋β)淀粉酶总活力的测定。

3.3 血清（浆）和唾液等液体样本

直接测定，建议预实验确定合适的稀释倍数，适当稀释的液体样本即为淀粉酶原液。吸取上述淀粉酶原液1mL，加入4mL去离子水，摇匀，即为淀粉酶稀释液，用于(α＋β)淀粉酶总活力的测定。注意：

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。

如需测定蛋白浓度，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、实验步骤

4.1 酶标仪或可见分光光度计预热30min以上，调节波长到540nm，可见分光光度计用去离子水调零；水浴锅预热到70℃。

4.2 每个样本取2支EP管分别加入75μL淀粉酶原液和淀粉酶稀释液，沸水浴5min，分别作为α-淀粉酶对照管和总淀粉酶对照管使用。

4.3 样本测定（在EP管中依次加入下列试剂）：

4.4 混匀，沸水浴5min，冷却，取200μL至96孔板或微量玻璃比色皿中，测定540nm处的吸光值，计算ΔA_α=A_测定α-A_对照α；ΔA_总=A_测定总-A_对照总；ΔA_标准=A_标准-A_空白。

注意：每个样本需分别设置α-淀粉酶活力测定对照和总淀粉酶活力测定对照，空白管只需做1管。实验之前建议选择2-3个预期差异大的样本做预实验。如果样本A_测定大于2，则说明样本酶活力太高，必须用去离子水稀释成适当浓度（计算公式中乘以相应稀释倍数）。若样本ΔA_测定小于0.005可以重新提取样本减少去离子水定容的体积。

5、结果计算

5.1 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_α带入方程得到y1值（mg/mL），ΔA_总带入方程得到y2值（mg/mL）。

5.2 α-淀粉酶活性计算

(1) 按样本质量计算

单位定义：每g组织每分钟催化产生1mg还原糖定义为1个酶活力单位。 α-淀粉酶活性 (U/g 质量)=y1×V_样÷(W×V_样÷V_样总)÷T×n=2×y1÷W×n

(2) 按样本蛋白浓度计算

单位的定义：每mg组织蛋白每分钟产生1mg还原糖定义为1个酶活力单位。 α-淀粉酶活性 (U/mg prot)=y1×V_样÷(Cpr×V_样)÷T×n=0.2×y1÷Cpr×n (3) 按液体样本体积计算单位的定义：每L液体样本每分钟产生1mg还原糖定义为1个酶活力单位。 α-淀粉酶活性 (U/L)=1,000×y1÷T×n=200×y1×n

5.3 总淀粉酶活性计算

(1) 按照样本质量计算

单位定义：每g组织每分钟催化产生1mg还原糖定义为1个酶活力单位。总淀粉酶活性 (U/g 质量)=5×y2×V_样÷(W×V_样÷V_样总)÷T×n=10×y2÷W×n

(2) 按照蛋白质含量计算

单位定义：每mg组织蛋白每分钟催化产生1mg还原糖定义为1个酶活力单位。总淀粉酶活性 (U/mg prot)=5×y2×V_样÷(V_样×Cpr)÷T×n=y2÷Cpr×n

(3) 按液体样本体积计算

单位的定义：每L液体样本每分钟产生1mg还原糖定义为1个酶活力单位。总淀粉酶活性 (U/L)=5×1,000×y2÷T×n=1,000×y2×n

5.4 β-淀粉酶活性计算

(1) 按照样本质量计算

单位定义：每g组织在反应体系中每分钟催化产生1mg还原糖定义为1个酶活力单位。 β-淀粉酶活性 (U/g 质量)=淀粉酶总活性-α-淀粉酶活性=(10×y2÷W×n)-(2×y1÷W×n)=(10×y2-2×y1)÷W×n

(2) 按照蛋白质含量计算

单位定义：每mg组织蛋白在反应体系中每分钟催化产生1mg还原糖定义为1个酶活力单位。 β-淀粉酶活性 (U/mg prot)=淀粉酶总活性-α-淀粉酶活性=(y2÷Cpr×n)-(0.2×y1÷Cpr×n)=(y2-0.2×y1)÷Cpr×n

(3) 按液体样本体积计算

单位的定义：每L液体样本每分钟产生1mg还原糖定义为1个酶活力单位。 β-淀粉酶活性 (U/L)=淀粉酶总活性-α-淀粉酶活性=(1,000×y2×n)-(200×y1×n)=(1,000×y2-200×y1)×n

参数说明：

V_样：加入反应体系中样本体积，0.075 mL；

W：样本质量，g；

V_样总：样本总体积，10mL；

T：反应时间，5min；

n：样本稀释倍数；

Cpr：样本蛋白质浓度，mg/mL；

1,000：1 L=1,000 mL；

5：总淀粉酶稀释倍数。

6、结果展示

典型标准曲线：y=0.4948x-0.0179，R²=0.9982

注意事项

本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。

Amylase is responsible for hydrolyzing starch and mainly includes α-amylase (α-AL) and β-amylase (β-AL, EC 3.2.1.2). β-amylase cleaves α-1,4 glycosidic bonds from the non-reducing ends of starch, producing reducing sugars such as glucose, maltose, maltotriose, and dextrin.

Detection Principle

Amylase catalyzes the hydrolysis of starch to generate reducing sugars. The reducing sugars then reduce 3,5-dinitrosalicylic acid to form a brown-red product with an absorption peak at 540 nm. The amylase activity is calculated by measuring the rate of absorbance increase at 540 nm. α-Amylase is heat-resistant, while β-amylase can be inactivated by incubation at 70°C for 15 minutes. Therefore, after treating the crude enzyme extract at 70°C for 15 min, only α-amylase can catalyze starch hydrolysis. Total amylase activity is measured using the untreated crude enzyme extract. β-Amylase activity is obtained by subtracting the α-amylase activity from the total amylase activity.

Detection Range: 0.0156-1 mg/mL

Sensitivity: 0.0078 mg/mL

Applicable Samples: Serum (plasma), Saliva, Animal and plant tissues

Note: Before formal testing, it is recommended to perform a preliminary experiment with 2-3 samples expected to show significant differences.

Reagents, consumables and Equipments not provided

Microplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)
96-well plate or micro-volume glass cuvette, Adjustable pipettes and tips
Low-temperature centrifuge, Water bath, Ice maker, Incubator
Deionized water
Homogenizer (for tissue samples)

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
DNS Reagent	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C protected from light.
Substrate	Prepare immediately before use. For 96T, add 42 mL deionized water; for 48T, add 21 mL deionized water. Invert several times and heat at 95°C until dissolved.	Dissolved Substrate can be stored at 4°C for one week. If precipitation occurs, reheat at 95°C to dissolve before use.
Standard	Contains 10 mg anhydrous glucose. Dissolve in 1 mL deionized water immediately before use to obtain a 10 mg/mL standard solution.	Dissolved standard can be stored at 4°C for two weeks.

2. Standard Curve Setup

Dilute the 10 mg/mL standard solution with deionized water as shown in the table below to obtain standard solutions of 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, and 0.0156 mg/mL.

Tube	Standard Volume	Deionized Water Volume (µL)	Standard Concentration (mg/mL)
Std.1	40µL 10mg/mL	360	1
Std.2	200µL of Std.1 (1mg/mL)	200	0.5
Std.3	200µL of Std.2 (0.5mg/mL)	200	0.25
Std.4	200µL of Std.3 (0.25mg/mL)	200	0.125
Std.5	200µL of Std.4 (0.125mg/mL)	200	0.0625
Std.6	200µL of Std.5 (0.0625mg/mL)	200	0.0313
Std.7	200µL of Std.6 (0.0313mg/mL)	200	0.0156

Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3. Sample Preparation

Note: Fresh samples are recommended.

3.1 Animal Tissue

Weigh approximately 0.1 g of sample, add 1 mL of deionized water, and homogenize. Transfer the homogenate to a centrifuge tube and let it stand at room temperature for 15 minutes, vortexing once every 5 minutes for thorough extraction. Centrifuge at 6,000 g, room temperature for 10 minutes. Aspirate the supernatant and dilute to 10 mL with deionized water. Mix well; this is the amylase stock solution. Take 1 mL of this amylase stock solution, add 4 mL of deionized water, and mix well; this is the amylase working solution, used for measuring total (α + β) amylase activity.

3.2 Plant Tissue

Weigh approximately 0.1 g of sample, add 1 mL of deionized water, and grind. Sonicate for 5 minutes (power 20% or 200 W, pulse 3 seconds, interval 7 seconds, repeat 30 times). Let it stand at room temperature for 15 minutes, vortexing once every 5 minutes for thorough extraction. Centrifuge at 6,000 g, room temperature for 10 minutes. Aspirate the supernatant and dilute to 10 mL with deionized water. Mix well; this is the amylase stock solution. Take 1 mL of this amylase stock solution, add 4 mL of deionized water, and mix well; this is the amylase working solution, used for measuring total (α + β) amylase activity.

3.3 Serum (Plasma), Saliva, and Other Liquid Samples

Test directly. It is recommended to perform a preliminary experiment to determine an appropriate dilution factor. The appropriately diluted liquid sample serves as the amylase stock solution. Take 1 mL of this amylase stock solution, add 4 mL of deionized water, and mix well; this is the amylase working solution, used for measuring total (α + β) amylase activity.

Note:

For animal tissues with high fat content, remove the upper fat layer after centrifugation before taking the supernatant.
If protein concentration measurement is required, we recommend using the Aladdin B665595 BCA Protein Quantification Kit or the R1491648 Ready-to-use BCA Protein Quantification Kit.

4. Experimental Steps

4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes and set the wavelength to 540 nm. Zero the visible spectrophotometer with deionized water. Preheat a water bath to 70°C.

4.2 For each sample, take two EP tubes and add 75 µL of the amylase stock solution and 75 µL of the amylase working solution, respectively. Boil in a water bath for 5 minutes. These will serve as the α-amylase control tube and the total amylase control tube, respectively.

4.3 Sample Assay (Add the following reagents sequentially in EP tubes):

4.4 Mix well, boil in a water bath for 5 minutes, then cool. Transfer 200 µL to a 96-well plate or micro-volume glass cuvette. Measure the absorbance at 540 nm. Calculate ΔA_α = A_{Test α} - A_{Control α}; ΔA_Total = A_{Test Total} - A_{Control Total}; ΔA_Standard = A_Standard - A_Blank.

Note: Each sample requires separate controls for the α-amylase activity assay and the total amylase activity assay. Only one blank tube is needed. It is recommended to perform a preliminary experiment with 2-3 samples showing expected large differences before formal testing. If the sample A_Test is greater than 2, the sample enzyme activity is too high and must be diluted with deionized water to an appropriate concentration (multiply by the corresponding dilution factor in the calculation). If the sample ΔA_Test is less than 0.005, the sample can be re-extracted by reducing the volume of deionized water used for dilution.

5. Calculation of Results

5.1 Plotting the Standard Curve

5.1 Plotting the Standard Curve Plot the standard curve with the standard concentration as the y-axis and ΔA_Standard as the x-axis. Substitute ΔA_α into the equation to obtain the y1 value (mg/mL), and substitute ΔA_Total to obtain the y2 value (mg/mL).

5.2 α-Amylase Activity Calculation

(1) Based on sample mass Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per gram of tissue. α-Amylase Activity (U/g mass) = y1 × V_Sample ÷ (W × V_Sample ÷ V_{Total Extract}) ÷ T × n = 2 × y1 ÷ W × n

(2) Based on sample protein concentration Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per mg of tissue protein. α-Amylase Activity (U/mg prot) = y1 × V_Sample ÷ (Cpr × V_Sample) ÷ T × n = 0.2 × y1 ÷ Cpr × n

(3) Based on liquid sample volume Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per liter of liquid sample. α-Amylase Activity (U/L) = 1,000 × y1 ÷ T × n = 200 × y1 × n

5.3 Total Amylase Activity Calculation

(1) Based on sample mass

Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per gram of tissue. Total Amylase Activity (U/g mass) = 5 × y2 × V_Sample ÷ (W × V_Sample ÷ V_{Total Extract}) ÷ T × n = 10 × y2 ÷ W × n

(2) Based on protein content

Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per mg of tissue protein. Total Amylase Activity (U/mg prot) = 5 × y2 × V_Sample ÷ (V_Sample × Cpr) ÷ T × n = y2 ÷ Cpr × n

(3) Based on liquid sample volume

Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per liter of liquid sample. Total Amylase Activity (U/L) = 5 × 1,000 × y2 ÷ T × n = 1,000 × y2 × n

5.4 β-Amylase Activity Calculation

(1) Based on sample mass

Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per gram of tissue in the reaction system. β-Amylase Activity (U/g mass) = Total Amylase Activity - α-Amylase Activity = (10 × y2 ÷ W × n) - (2 × y1 ÷ W × n) = (10 × y2 - 2 × y1) ÷ W × n

(2) Based on protein content

(2) Based on protein content Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per mg of tissue protein in the reaction system. β-Amylase Activity (U/mg prot) = Total Amylase Activity - α-Amylase Activity = (y2 ÷ Cpr × n) - (0.2 × y1 ÷ Cpr × n) = (y2 - 0.2 × y1) ÷ Cpr × n

(3) Based on liquid sample volume

(3) Based on liquid sample volume Unit definition: One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per liter of liquid sample. β-Amylase Activity (U/L) = Total Amylase Activity - α-Amylase Activity = (1,000 × y2 × n) - (200 × y1 × n) = (1,000 × y2 - 200 × y1) × n

Parameter Description:

V_Sample: Volume of sample added to the reaction system, 0.075 mL;

W: Sample mass, g;

V_{Total Extract}: Total volume of the sample extract, 10 mL;

T: Reaction time, 5 min;

n: Sample dilution factor;

Cpr: Sample protein concentration, mg/mL;

1,000: 1 L = 1,000 mL;

5: Dilution factor for total amylase.

6. Example Results

Typical Standard Curve: y = 0.4948x - 0.0179, R² = 0.9982

Notes

This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

规格

别名

β-淀粉酶活性检测试剂盒 | β-淀粉酶活性检测试剂盒微量法 | β-淀粉酶活性检测试剂盒（3,5-二硝基水杨酸法）

英文别名

β-Amylase (β-AL) Activity Assay Kit

规格或纯度

BioReagent

英文名称

β-Amylase (β-AL) Activity Assay Kit (DNS, Micro Method)

应用

细胞代谢

样品类型

动植物组织, 唾液, 血浆, 血清

检测范围

0.0156-1 mg/mL

检测方法

比色法

检测仪器

酶标仪,紫外-可见光分光光度计

检测波长

540

敏感性

Light-sensitive

储存与运输

储存条件

2-8°C储存,避光

运输条件

低温运输

稳定性与储存

Store at 2-8℃ long term (12 months). Store in the dark.

组分与储存

A1508361	Component	48T	96T	Storage
A1508361A	DNS Reagent	40 mL	80 mL	2-8℃. Store in the dark.
A1508361B	Substrate	1EA	1EA	2-8℃
A1508361C	Standard	1EA	1EA	2-8℃

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批号(Lot Number)	证书类型	货号
ZJ26F0232334	分析证书	A1508361

技术文档和文章

a1508361 β 淀粉酶 β al 活性检测试剂盒 dns 微量法中文说明书.pdf

饲料与食品发酵中阿魏酸酯酶的作用机制及应用

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半纤维素阿魏酸酯酶阿魏酸植物细胞壁饲料发酵食品发酵酚酸释放复合酶协同

Mechanisms and Applications of Feruloyl Esterase in Feed and Food Fermentation

目录：技术文章

探索主题:

Hemicellulose feruloyl esterase ferulic acid Plant Cell Wall Feed Fermentation Food Fermentation Phenolic Acid Release Multienzyme Synergy

溶液计算器

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稀释计算器

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复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

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所需的复溶浓度

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β-淀粉酶 (β-AL) 活性检测试剂盒（DNS, 微量法）

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