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

Cat. No.: A1508361

GRADE & PURITY BioReagent

Overview

  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.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

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

Contents & Storage

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℃