BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Store at 2-8°C,Protected from light Ships Wet ice Check lot-specific COA for exact specifications.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Starch hydrolases include α-amylase (α-AL, EC 3.2.1.1) and β-amylase (β-AL). α-Amylase randomly catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch, producing reducing sugars such as glucose, maltose, maltotriose, and dextrins, while simultaneously reducing the viscosity of starch, hence it is also known as the liquefying enzyme. α-Amylase is widely distributed, from microorganisms to higher plants.
Detection Principle: Starch hydrolases catalyze the hydrolysis of starch to produce reducing sugars. These reducing sugars reduce 3,5-dinitrosalicylic acid (DNS) to produce a brown-red-colored compound with an absorption peak at 540 nm. The amylase activity is calculated by measuring the rate of increase in absorbance at 540 nm. α-Amylase is heat-stable, but β-amylase can be inactivated by heating at 70°C for 15 minutes. Therefore, after the crude enzyme extract is treated at 70°C for 15 minutes, only α-amylase can catalyze starch hydrolysis.
Detection Range: 0.0156 - 1 mg/mL
Sensitivity: 0.0078 mg/mL
Applicable Samples: Saliva, animal tissues, plant tissues (seeds or newly germinated seedlings)
Note: The detection range and sensitivity are based on the standard. The actual detection range and sensitivity for activity need to be calculated according to the sample conditions.
| G1501772 | Component | 96T | Storage |
| G1501772A | DNS Reagent | 40 mL | 2-8℃. Store in the dark. |
| G1501772B | Substrate | 1EA | 2-8℃ |
| G1501772C | Standard | 1EA | 2-8℃ |
Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
User-Prepared Instruments and Reagents
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Notes |
| DNS Reagent | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Substrate | Before use, add 20 mL deionized water, invert and shake several times, heat until dissolved. | Unused reagent can be stored at 4°C for one week. If precipitate forms, heat to 70°C to dissolve. |
| Standard | Before use, add 1 mL deionized water to dissolve, obtaining a 10 mg/mL standard (Glucose) stock. | Can be stored at 4°C for 2 weeks. |
2. Standard Curve Setup
Dilute the 10 mg/mL standard stock solution with deionized water to concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, and 0.0156 mg/mL as shown in the table below.
| Tube | Standard Volume | Deionized Water Volume (μL) | Standard Concentration (mg/mL) |
| Std.1 | 40μL (10 mg/mL) | 360 | 1 |
| Std.2 | 200μL of Std.1 | 200 | 0.5 |
| Std.3 | 200μL of Std.2 | 200 | 0.25 |
| Std.4 | 200μL of Std.3 | 200 | 0.125 |
| Std.5 | 200μL of Std.4 | 200 | 0.0625 |
| Std.6 | 200μL of Std.5 | 200 | 0.0313 |
| Std.7 | 200μL of Std.6 | 200 | 0.0156 |
Note: The standard curve must be generated with 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 tissue. Add 1 mL of deionized water and homogenize. Transfer the homogenate to a centrifuge tube. Let it stand at room temperature for 15 minutes, vortexing every 5 minutes for sufficient extraction. Centrifuge at 6,000 g for 10 minutes at room temperature. Aspirate the supernatant and dilute to 10 mL with deionized water. Mix well. This is the amylase stock solution.
3.2 Plant Tissue
Weigh approximately 0.1 g of tissue. Add 1 mL of deionized water and grind. Sonicate for 5 minutes (power 20%, pulse 3s on, 7s off, repeat 30 times). Let it stand at room temperature for 15 minutes, vortexing every 5 minutes for sufficient extraction. Centrifuge at 6,000 g for 10 minutes at room temperature. Aspirate the supernatant and dilute to 10 mL with deionized water. Mix well. This is the amylase stock solution.
3.3 Saliva, and Other Liquid Samples
Assay directly. It is recommended to perform a preliminary test to determine the appropriate dilution factor.
Note:
For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.
If protein concentration measurement is required, use Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648).
4. Assay Steps
4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 540 nm. For spectrophotometers, zero the instrument with deionized water.
4.2 Preheat a water bath to 70°C.
4.3 Take 75 μL of sample and incubate in a boiling water bath for 5 minutes. This will be used as the Control tube.
4.4 Sample Measurement (Add reagents sequentially into microcentrifuge tubes as below):
| Reagent | Blank Tube (μL) | Standard Tube (μL) | Test Tube (μL) | Control Tube (μL) |
| Deionized Water | 75 | 0 | 0 | 0 |
| Standard (various conc.) | 0 | 75 | 0 | 0 |
| Sample | 0 | 0 | 75 | 75 (boiled sample) |
Heat at 70°C for 15 min, then cool.
| Substrate | 0 | 0 | 75 | 0 |
Incubate in a constant temperature water bath at 40°C for 5 min.
| DNS Reagent | 150 | 150 | 150 | 150 |
| Substrate | 75 | 75 | 0 | 75 |
4.5 Mix well. Incubate in a boiling water bath for 5 minutes. Cool. Transfer 200 μL to a 96-well plate or micro glass cuvette. Measure the absorbance at 540 nm. Calculate ΔA test = A test - A control ; ΔA standard = A standard - A blank .
Note:
Each sample requires a control tube. The blank tube only needs to be prepared once.
It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If A test > 2, the enzyme activity is too high, and the sample must be diluted with deionized water to an appropriate concentration (multiply by the dilution factor in the calculation). If ΔA test < 0.005, re-extract the sample reducing the final volume of deionized water used for dilution.
5. Calculation of Results
5.1 Standard Curve Plotting Plot the standard concentration (y-axis) against ΔA standard (x-axis) to generate the standard curve. Substitute ΔA test into the standard curve equation to calculate y (mg/mL).
5.2 α-Amylase Activity Calculation
(1) Based on Sample Fresh Weight Calculation
(1) Based on Sample Fresh Weight Calculation Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per gram of tissue. Calculation Formula: α-Amylase Activity (U/g weight) = y × V sample ÷ (W × V sample ÷ V total ) ÷ T × n = 2 × y ÷ W × n
(2) Based on Sample Protein Concentration
(2) Based on Sample Protein Concentration Calculation Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per milligram of tissue protein. Calculation Formula: α-Amylase Activity (U/mg prot) = y × V sample ÷ (Cpr × V sample ) ÷ T × n = 0.2 × y ÷ Cpr × n
(3) Based on Liquid Sample Volume
Calculation Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per liter of liquid sample. Calculation Formula:
α-Amylase Activity (U/L) = 1000 × y ÷ T × n = 200 × y × n
Parameter Definitions:
y: Concentration of reducing sugar calculated from the standard curve (mg/mL)
V sample : Volume of sample added to the reaction system (0.075 mL)
W: Sample weight (g)
V total : Total volume of the sample extract (10 mL)
T: Enzymatic reaction time (5 minutes)
n: Sample dilution factor
Cpr: Sample protein concentration (mg/mL)
1000: Conversion factor between liters and milliliters (1 L = 1000 mL)
6. Representative Results
Typical Standard Curve: y = 0.4948x - 0.0179, R² = 0.9982
Precautions
1. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, please use appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.
2. This product is for research use only. Not for use in clinical diagnosis.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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