β-Amylase(β-AL) Activity Assay Kit
Spectrophotometry
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ใช้สำหรับวัดกิจกรรมของเอนไซม์ β-Amylase ในตัวอย่างทางชีวภาพต่างๆ เช่น สารสกัดจากพืช ผลิตภัณฑ์อาหาร และบำรุงเลี้ยงจุลินทรีย์ ช่วยในการประเมินกระบวนการย่อยสลายแป้ง ซึ่งมีความสำคัญในอุตสาหกรรม เช่น การต้มเบียร์ การอบ และการผลิตเชื้อเพลิงชีวภาพ ทำให้สามารถควบคุมคุณภาพในกระบวนการแปรรูปอาหารโดยการตรวจสอบการแปลงแป้งเป็นมอลโตส สนับสนุนการวิจัยในสรีรวิทยาพืชและชีวเคมีเพื่อเข้าใจการทำงานของเอนไซม์ในเมแทบอลิซึมแป้ง นอกจากนี้ยังนำไปใช้ในการศึกษาทางเกษตรเพื่อปรับปรุงผลผลิตพืชและปริมาณแป้ง จัดหาเครื่องมือที่เชื่อถือได้สำหรับการศึกษากิเนติกส์เอนไซม์และการปรับปรุงกระบวนการเอนไซมัติในอุตสาหกรรม
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| พารามิเตอร์การทดสอบ | ข้อมูลจำเพาะ |
|---|---|
| Performance test | PASS |
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β-Amylase (β-AL) Activity Assay Kit
Product Information
| Product Code | 111983 |
|---|---|
| Method | Spectrophotometry |
| Detection Wavelength | 540 nm |
| Kit Size | 50T |
Product Introduction
Amylases hydrolyze starch and mainly include α-amylase and β-amylase. β-Amylase (EC 3.2.1.2) acts on α-1,4-glycosidic bonds in starch and produces glucose, maltose, maltotriose, dextrin, and other reducing sugars.
Reducing sugars reduce 3,5-dinitrosalicylic acid to form a reddish-brown substance. α-Amylase is not acid-resistant, and β-amylase is not heat-resistant. Based on these characteristics, one type of amylase can be measured by inactivating the other.
Example test sample: sweet potato. OD540 nm readings: α-amylase assay 1.843/1.879, control 0.858/0.871; total amylase assay 1.406/1.465, control 0.347/0.351. Actual readings may vary depending on the detection instrument and test conditions. These data are for reference only.
Package Contents
| Component Code | Component | Quantity |
|---|---|---|
| BR5000020.1 | Reagent One | 50 mL |
| BR5000020.2 | Reagent Two | 30 mL |
| BR5000020.3 | Standard | 5 mg |
| BR5000020.m | Instruction Manual | 1 copy |
Quality and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent One | -- | -- |
| Reagent Two | -- | -- |
| Standard | -- | -- |
Transportation and Storage
| Transportation | Transport with ice packs. |
|---|---|
| Storage | Store Reagent I at 2-8°C protected from light. Store Reagent II at room temperature. |
| Shelf Life | 180 days |
Instructions for Use
1. Preparation of Crude Enzyme Extract
Tissue Samples
- Weigh 0.1-0.2 g sample. Approximately 0.1 g is recommended.
- Add 1 mL distilled water and grind to homogenize.
- Transfer the homogenate into a centrifuge tube and allow it to extract at room temperature for 15 min. Vortex once every 5 min to ensure full extraction.
- Centrifuge at 3000 g and 25°C for 10 min.
- Collect the supernatant and dilute to 10 mL with distilled water. Mix well. This is the amylase stock solution.
- Pipette 1 mL amylase stock solution, add 4 mL distilled water, and mix well to obtain the diluted amylase solution for total (α + β) amylase activity determination.
Serum, Plasma, and Other Liquid Samples
- Use the amylase stock solution directly for the α-amylase assay.
- Pipette 1 mL amylase stock solution, add 4 mL distilled water, and mix well to obtain the diluted amylase solution for total (α + β) amylase activity determination.
2. Solution Preparation
Before use, add 1 mL distilled water to the Standard and dissolve to prepare a 5 mg/mL standard solution. The prepared standard solution can be stored at 2-8°C for one week.
3. Assay Preparation
- Preheat the spectrophotometer for at least 30 min. Set the wavelength to 540 nm and zero with distilled water.
- Preheat Reagent 1 and Reagent 2 at 40°C for 10 min.
- Dilute the 5 mg/mL standard solution with distilled water to prepare 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL standard solutions for testing.
Standard Solution Dilution
| No. | Concentration Before Dilution (mg/mL) | Standard Solution Volume (µL) | Distilled Water Volume (µL) | Concentration After Dilution (mg/mL) |
|---|---|---|---|---|
| 1 | 5 | 100 | 900 | 0.5 |
| 2 | 0.5 | 500 | 500 | 0.25 |
| 3 | 0.25 | 500 | 500 | 0.125 |
| 4 | 0.125 | 500 | 500 | 0.0625 |
| 5 | 0.0625 | 500 | 500 | 0.03125 |
In the assay below, each standard tube requires 250 µL standard solution. Do not measure absorbance directly at this dilution step.
4. Control Preparation
Prepare two tubes. Add 250 µL amylase stock solution to one tube and 250 µL amylase dilution solution to the other tube. Boil both tubes in a water bath for 30 min. Use them as the α-amylase control tube and total amylase control tube, respectively.
5. Assay Procedure
| Component or Step | α-Amylase Control Tube | α-Amylase Assay Tube | Total Amylase Control Tube | Total Amylase Assay Tube | Blank Tube | Standard Tube |
|---|---|---|---|---|---|---|
| Amylase stock solution (µL) | 250, boiled | 250 | ||||
| Distilled water (µL) | 250 | |||||
| Standard solution (µL) | 250 | |||||
| Water bath | 70°C for approximately 15 min, then cool quickly on ice | 70°C for approximately 15 min, then cool quickly on ice | ||||
| Amylase dilution solution (µL) | 250, boiled | 250 | ||||
| Reagent II (µL) | 250 | 250 | 250 | 250 | ||
| Incubation | 40°C constant-temperature water bath for exactly 10 min | 40°C constant-temperature water bath for exactly 10 min | 40°C constant-temperature water bath for exactly 10 min | 40°C constant-temperature water bath for exactly 10 min | ||
| Reagent II (µL) | 250 | 250 | ||||
| Reagent I (µL) | 500 | 500 | 500 | 500 | 500 | 500 |
| Color development and reading | Mix well, heat in a 95°C water bath for 10 min, cool, and read absorbance at 540 nm. | |||||
Record the absorbance values from left to right as A1, A2, A3, A4, A5, and A6.
Calculate: ΔAα = A2 - A1; ΔAtotal = A4 - A3; ΔAstandard = A6 - A5.
Each assay tube requires one control tube. The blank tube and standard curve only need to be measured once or twice.
Activity Calculation
1. Standard Curve
Use the standard tube concentration (X, mg/mL) and absorbance ΔAstandard (Y, ΔAstandard) to establish the standard curve. Substitute ΔAα (Y, ΔA) into the standard curve formula to calculate sample concentration X1 (mg/mL). Substitute ΔAtotal into the formula to calculate sample concentration X2 (mg/mL).
2. α-Amylase Activity
Calculated by sample mass: each g of tissue catalyzing the production of 1 mg reducing sugar per minute is defined as 1 enzyme activity unit.
α-Amylase activity (mg/min/g fresh weight) = X1 × Vtotal reaction ÷ (W × Vsample ÷ Vtotal sample) ÷ T
Calculated by protein content: each mg of tissue protein catalyzing the production of 1 mg reducing sugar is defined as 1 enzyme activity unit.
α-Amylase activity (mg/min/mg protein) = X1 × Vtotal reaction ÷ (Vsample × Cpr) ÷ T
For serum, plasma, and other liquid samples: each mL serum or plasma catalyzing the production of 1 mg reducing sugar per minute is defined as 1 enzyme activity unit.
α-Amylase activity (mg/min/mL) = X1 × Vtotal reaction ÷ Vsample ÷ T
3. Total Amylase Activity
Calculated by sample mass: each g of tissue catalyzing the production of 1 mg reducing sugar per minute is defined as 1 enzyme activity unit.
Total amylase activity (mg/min/g fresh weight) = 5 × X2 × Vtotal reaction ÷ (W × Vsample ÷ Vsample total) ÷ T
Calculated by protein content: each mg of tissue protein catalyzing the production of 1 mg reducing sugar per minute is defined as 1 enzyme activity unit.
Total amylase activity (mg/min/mg protein) = 5 × X2 × Vtotal reaction ÷ (Vsample × Cpr) ÷ T
For serum, plasma, and other liquid samples: each mL serum or plasma catalyzing the production of 1 mg reducing sugar per minute is defined as 1 enzyme activity unit.
Total amylase activity (mg/min/mL) = 5 × X2 × Vreaction total ÷ Vsample ÷ T
4. β-Amylase Activity
β-Amylase activity (mg/min/g fresh weight) = total amylase activity - α-amylase activity
Formula Parameters
| Symbol | Description |
|---|---|
| 5 | Total amylase dilution factor |
| Vreaction total | Total volume of the reaction system, 0.5 mL |
| Vsample | Sample volume added to the reaction system, 0.25 mL |
| Vsample total | Total volume of extract, 10 mL |
| Cpr | Sample protein concentration, mg/mL |
| W | Sample mass, g |
| T | Reaction time, 10 min |
Notes
- Before formal measurement, select 2-3 samples with large expected differences for preliminary testing. This 50T reagent kit can test 24 samples.
- Required instruments and supplies: visible spectrophotometer, analytical balance, homogenizer or mortar, refrigerated centrifuge, water bath, adjustable pipette, 1 mL glass cuvette, ice, and distilled water.
- If yellow crystals precipitate from Reagent I, heat at 60°C to dissolve before use.
- If Reagent II shows precipitate, heat at 70°C to dissolve before use.
- The linear range of this kit is 0.03125-0.5 mg/mL.
- If the measured absorbance value is greater than 2, dilute the sample appropriately before measurement. If the absorbance value is too low, concentrate the amylase diluent or the original amylase solution.
- This product is for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, must not be used in food or drugs, and must not be stored in ordinary residences.
- For safety and health, wear a lab coat and disposable gloves during operation.
Appendix
For greater accuracy, prepare a standard curve for each experiment. Use the procedure table above to obtain absorbance values for each standard tube, plot the standard curve with R2≥ 0.99, and use the resulting calculation formula for sample calculation.
Visual Reference
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