Solid-Cellulase(S-CL) Activity Assay Kit
enzyme labeling method
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description Product Description
The Solid-Cellulase (S-CL) Activity Assay Kit is primarily used to measure the enzymatic activity of cellulase in various samples. This kit is essential in industries such as biofuel production, where cellulase enzymes break down cellulose into fermentable sugars, a critical step in the conversion of biomass to ethanol. It is also widely applied in the textile industry for bio-polishing and stone-washing processes, where cellulase helps in softening fabrics and creating desired finishes. Additionally, the kit is utilized in the paper and pulp industry to improve the quality of paper by modifying cellulose fibers. In research and development, it serves as a valuable tool for studying enzyme kinetics and optimizing cellulase production in microbial strains. The kit provides a reliable and efficient method for quantifying cellulase activity, ensuring consistent results in industrial and laboratory settings.
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| Test Parameter | Specification |
|---|---|
| Performance test | PASS |
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Solid-Cellulase (S-CL) Activity Assay Kit, Microplate Method
Product code: 67128
This kit measures soil cellulase (S-CL) activity. S-CL is mainly derived from soil microorganisms and catalyzes cellulose in crop straw to produce glucose, an important carbon source nutrient. The assay uses the 3,5-dinitrosalicylic acid method to determine S-CL activity based on the reducing sugars produced during cellulose degradation.
Package Contents and Storage
| Code | Item | Quantity | Storage |
|---|---|---|---|
| 67128.1 | Reagent I | Self-prepared | / |
| 67128.2 | Reagent II | 6 mL | 2-8°C |
| 67128.3 | Reagent III | 25 mL | 2-8°C |
| 67128.4 | Reagent IV | 3.5 mL | 2-8°C, protected from light |
| 67128.5 | Standard | 10 mg | 2-8°C |
| 67128.m | Instructions | 1 copy | / |
Quality and Safety Information
| Material | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | -- | -- |
| Reagent II | -- | -- |
| Reagent III | -- | -- |
| Reagent IV | -- | -- |
| Standard | -- | -- |
Transportation and Storage
Transportation: Transport with ice packs.
Storage: Store according to the conditions listed above. Shelf life is 180 days.
Materials Required but Not Provided
- Microplate reader
- Water bath or metal bath
- Adjustable pipettor
- 96-well plate
- 30-50 mesh sieve
- Toluene
- Distilled water
Instructions for Use
1. Sample Processing
Naturally air-dry fresh soil samples, or dry them in a 37°C oven. Pass the dried samples through a 30-50 mesh sieve.
2. Reagent Preparation
- Reagent I: Toluene, 3 mL × 1 bottle. Prepare by the user.
- Standard: Add 1 mL distilled water to 10 mg anhydrous glucose and dissolve to prepare a 10 mg/mL glucose solution. Store at 2-8°C for up to two weeks, or dissolve with saturated benzoic acid solution for longer storage.
3. Assay Procedure
- Preheat the microplate reader for 30 min. Set the wavelength to 540 nm and zero with distilled water.
- Dilute the 10 mg/mL standard solution with distilled water to prepare 1, 0.8, 0.6, 0.4, and 0.2 mg/mL standard solutions.
Standard Solution Dilution
| No. | Concentration Before Dilution (mg/mL) | Standard Solution Volume (µL) | Distilled Water Volume (µL) | Concentration After Dilution (mg/mL) |
|---|---|---|---|---|
| 1 | 10 | 100 | 900 | 1 |
| 2 | 1 | 160 | 40 | 0.8 |
| 3 | 1 | 120 | 80 | 0.6 |
| 4 | 1 | 80 | 120 | 0.4 |
| 5 | 1 | 40 | 160 | 0.2 |
In the following experiment, each standard tube requires 10 µL standard solution. Do not measure absorbance directly at this dilution step.
Assay Setup
| Component or Step | Control Tube | Assay Tube | Standard Tube | Blank Tube |
|---|---|---|---|---|
| Air-dried soil sample (g) | 0.05 | 0.05 | - | - |
| Reagent I (µL) | 25 | 25 | - | - |
| Control pretreatment | Boil for 15 min. Wrap with sealing film to prevent the cap from popping off. | Shake and mix thoroughly; let stand at room temperature for 15 min. | - | - |
| Reagent II (µL) | 50 | 50 | - | - |
| Reagent III (µL) | 200 | 200 | - | - |
| Distilled water (µL) | 50 | 50 | - | - |
| Saccharification | Shake and mix thoroughly. Incubate in a 40°C water bath for 1 h, then boil for 15 min. Wrap with sealing film to prevent caps from popping. Cool, centrifuge at 10000 rpm at room temperature for 10 min, and collect the supernatant as the saccharification solution. | - | - | |
| Saccharification solution (µL) | 10 | 10 | - | - |
| Standard (µL) | - | - | 10 | - |
| Distilled water (µL) | - | - | - | 10 |
| Reagent IV (µL) | 30 | 30 | 30 | 30 |
| Color development | Mix thoroughly and boil in a boiling water bath for 15 min. Wrap with sealing film to prevent caps from popping. Cool. | |||
| Distilled water (µL) | 210 | 210 | 210 | 210 |
| Measurement | Mix thoroughly. After cooling, transfer 200 µL to a 96-well plate and measure absorbance at 540 nm. | |||
Record absorbance values as Acontrol, Aassay, Astandard, and Ablank.
Calculate: ΔAassay= Aassay- Acontrol
Calculate: ΔAstandard= Astandard- Ablank
Set one control tube for each assay tube. Blank tubes and the standard curve only need to be run 1-2 times.
Activity Calculation
1. Standard Curve
Use the standard tube concentration (X, mg/mL) and absorbance ΔAstandard(Y) to establish the standard curve. Substitute ΔAassay(Y) into the standard curve equation to calculate the sample concentration (X, mg/mL).
2. S-CL Enzyme Activity
Unit definition: The production of 1 mg glucose per g soil sample per day is defined as one enzyme activity unit.
S-CL enzyme activity (U/g soil sample) = X × Vtotal reaction÷ W ÷ T = 156 × X
- X: Sample concentration calculated from the standard curve, mg/mL
- T: Reaction time, 1 h = 1/24 d
- Vtotal reaction: Total volume of the reaction system, 0.325 mL
- W: Sample mass, 0.05 g
Precautions
- This 100T kit can test 48 samples. Before formal measurement, it is recommended to select 2-3 samples with large expected differences for a preliminary test.
- If the absorbance of the sample assay tube is too low, such as 0.01, the reaction time can be extended by increasing the 40°C water-bath saccharification time, possibly to 24 h or longer. The formula must be changed accordingly during calculation.
- The volume of saccharification solution used in the color development step can also be adjusted while reducing the volume of distilled water. In some cases, the entire distilled water volume may be replaced by saccharification solution. The standard curve must be modified accordingly.
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