β-Glucosidase(β-GC) Activity Assay Kit
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| Test Parameter | Specification |
|---|---|
| Performance test | PASS |
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β-Glucosidase (β-GC) Activity Assay Kit - Microplate Method
Product Information
| Product Code | 111992 |
| Assay Size | 100T |
| Detection Method | Microplate method |
Product Introduction
β-Glucosidase (β-GC, EC 3.2.1.21) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the hydrolysis of β-glycosidic bonds and has multiple physiological functions.
During cellulose saccharification, β-GC further hydrolyzes cellobiose and cello-oligosaccharides to produce glucose. β-GC also hydrolyzes terpene aroma precursors, converting glycosidically bound forms into free forms and producing aroma. In plants, β-GC can hydrolyze prunasin and release HCN, helping prevent insect feeding.
β-GC decomposes p-nitrophenyl-β-D-glucopyranoside to produce p-nitrophenol, which has a maximum absorption peak at 400 nm. β-GC activity is calculated by measuring the rate of increase in absorbance.
Product Packing List
| Code | Component | Quantity |
|---|---|---|
| 111992.1 | Reagent I | 1 bottle |
| 111992.2 | Reagent II | 1 bottle |
| 111992.3 | Reagent III | 1 bottle |
| 111992.4 | Extraction Solution | 1 bottle |
| 111992.m | Instruction Manual | 1 copy |
Quality Standards and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | -- | -- |
| Reagent II | -- | -- |
| Reagent III | -- | -- |
| Extraction Solution | -- | -- |
Transportation and Storage
| Transportation | Transport with ice packs. |
| Storage | Store Reagent I at -20°C. Store the remaining components at 2-8°C. |
| Shelf Life | 180 days |
Instructions for Use
1. Preparation of Crude Enzyme Extract
1.1 Bacteria or Cultured Cells
- Collect the bacteria or cells into a centrifuge tube.
- Centrifuge and discard the supernatant.
- Add Extraction Solution according to the ratio of bacteria or cells (104cells) to Extraction Solution volume (mL) at 500-1000:1. The recommended ratio is 5 million bacteria or cells with 1 mL Extraction Solution.
- Ultrasonically disrupt the bacteria or cells in an ice bath using 20% power or 200 W, ultrasound for 3 s, interval for 10 s, repeated 30 times.
- Centrifuge at 15000 g and 4°C for 10 min.
- Collect the supernatant and keep it on ice for testing.
1.2 Tissue
- Add Extraction Solution according to the ratio of tissue mass (g) to Extraction Solution volume (mL) at 1:5-10. It is recommended to weigh approximately 0.1 g tissue and add 1 mL Extraction Solution.
- Homogenize in an ice bath.
- Centrifuge at 15000 g and 4°C for 10 min.
- Collect the supernatant and keep it on ice for testing.
1.3 Liquid Samples
Culture medium, serum, plasma, and other liquid samples can be tested directly.
2. Reagent Preparation
Before use, add 12 mL distilled water to each bottle of Reagent I and dissolve thoroughly. Store unused prepared Reagent I at -20°C.
3. Assay Procedure
- Preheat the microplate reader for at least 30 min.
- Set the wavelength to 400 nm and zero with distilled water.
- Add reagents according to the table below.
| Component | Assay Tube | Control Tube |
|---|---|---|
| Reagent I (μL) | 120 | |
| Distilled water (μL) | 120 | |
| Reagent II (μL) | 150 | 150 |
| Sample (μL) | 30 | 30 |
- Mix thoroughly and place in a 37°C water bath for exactly 30 min.
- Immediately place in a 95°C water bath for 5 min. Cap tightly to prevent water loss.
- Cool under running water and mix thoroughly to ensure the concentration remains unchanged.
- Centrifuge at 8000 g and 4°C for 5 min.
- Collect the supernatant and add the following reagents to a 96-well plate.
| Component | Assay Tube | Control Tube |
|---|---|---|
| Supernatant (μL) | 70 | 70 |
| Reagent III (μL) | 130 | 130 |
- Mix thoroughly and let stand at room temperature for 2 min.
- Measure the absorbance at 400 nm as A.
- Calculate ΔA = Aassay- Acontrol.
Each assay tube requires one control tube.
β-GC Activity Calculation
The regression equation measured under standard conditions is y = 0.0039x - 0.0027, where x is the standard concentration (nmol/mL) and y is the absorbance value.
4.1 Calculation by Liquid Volume
Unit definition: the production of 1 nmol p-nitrophenol per mL sample per minute is defined as one unit of enzyme activity.
β-GC activity (nmol/min/mL) = [(ΔA + 0.0027) ÷ 0.0039 × Vtotal reaction] ÷ Vsample÷ T = 85.47 × (ΔA + 0.0027)
4.2 Calculation by Sample Protein Concentration
Unit definition: the production of 1 nmol p-nitrophenol per mg tissue protein per minute is defined as one unit of enzyme activity.
β-GC activity (nmol/min/mg prot) = [(ΔA + 0.0027) ÷ 0.0039 × Vtotal reaction] ÷ (Vsample× Cpr) ÷ T = 85.47 × (ΔA + 0.0027) ÷ Cpr
4.3 Calculation by Sample Fresh Weight
Unit definition: the production of 1 nmol p-nitrophenol per g tissue per minute is defined as one unit of enzyme activity.
β-GC activity (nmol/min/g, fresh weight) = [(ΔA + 0.0027) ÷ 0.0039 × Vtotal reaction] ÷ (W × Vsample÷ Vsample total) ÷ T = 85.47 × (ΔA + 0.0027) ÷ W
4.4 Calculation by Bacterial or Cell Density
Unit definition: the production of 1 nmol p-nitrophenol per 104bacteria or cells per minute is defined as one unit of enzyme activity.
β-GC activity (nmol/min/104cells) = [(ΔA + 0.0027) ÷ 0.0039 × Vreaction total] ÷ (500 × Vsample÷ Vsample total) ÷ T = 0.171 × (ΔA + 0.0027)
Formula Parameters
| Parameter | Description |
|---|---|
| Vreaction total | Total volume of the reaction system, 0.3 mL |
| Vsample | Sample volume added to the reaction system, 0.03 mL |
| Vsample total | Volume of Extraction Solution added, 1 mL |
| Cpr | Sample protein concentration, mg/mL |
| W | Sample mass, g |
| 500 | Total number of cells or bacteria, 5 million |
| T | Reaction time, 30 min |
Precautions
- This 100T kit can test 48 samples.
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