Alcohol Acyl Transferase(AAT) Activity Assay Kit
enzyme labeling method
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Alcohol Acyl Transferase (AAT) Activity Assay Kit is widely used in research and industrial applications to measure the enzymatic activity of AAT, which plays a crucial role in the biosynthesis of esters. These esters are key components in the flavor and fragrance industries, contributing to the aroma profiles of various food products, beverages, and perfumes. The kit is particularly valuable in studying the metabolic pathways involved in ester production, enabling the optimization of fermentation processes in winemaking, brewing, and dairy product development. Additionally, it aids in the characterization of enzymes for genetic engineering and biotechnological applications, helping to enhance the production of desired aromatic compounds. In plant biology, the kit is used to investigate the role of AAT in the synthesis of volatile organic compounds, which are essential for plant defense mechanisms and pollinator attraction. Overall, this assay kit is a vital tool for advancing research in biochemistry, food science, and biotechnology.
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Alcohol Acyl Transferase (AAT) Activity Assay Kit
Product code: 112885
Method: Microplate method
Product Introduction
Alcohol acyl transferase (AAT) belongs to a large family of multifunctional proteins that catalyze acylation and deacylation reactions in organisms. AAT plays important roles in gene expression, metabolism, and signal transduction.
AAT catalyzes the transfer of acetyl groups from acetyl-CoA to butanol. The released CoA reduces DTNB to generate TNB. TNB has an absorption peak at 412 nm, and the increase in absorbance at 412 nm can be used to calculate AAT activity.
Package Contents
| Code | Component | Quantity |
|---|---|---|
| 112885.1 | Reagent I | 1 bottle |
| 112885.2 | Reagent II | 1 bottle |
| 112885.3 | Reagent III | 1 bottle |
| 112885.4 | Extraction Solution | 1 bottle |
| 112885.m | Instruction Manual | 1 copy |
Quality and Safety Information
| Material | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | -- | -- |
| Reagent II | -- | -- |
| Reagent III | -- | -- |
| Extraction Solution | -- | -- |
Transportation and Storage
| Shipping | Shipped with ice packs. |
|---|---|
| Storage | Store Reagent II at -20°C. Store other components at 2-8°C protected from light. Shelf life: 180 days. |
Instructions for Use
1. Preparation of Crude Enzyme Extract
Tissue: Use a tissue mass (g) to Extraction Solution volume (mL) ratio of 1:5-10. It is recommended to weigh approximately 0.1 g tissue and add 1 mL Extraction Solution. Homogenize in an ice bath, then centrifuge at 8000g for 10 min at 4°C. Collect the supernatant and keep it on ice for testing.
Bacteria and fungi: Use a cell number (104cells) to Extraction Solution volume (mL) ratio of 500-1000:1. It is recommended to add 1 mL Extraction Solution to 5,000,000 cells. Disrupt cells by ultrasonic treatment in an ice bath at 300 W, with ultrasound for 3 seconds and an interval of 7 seconds, for a total time of 3 min. Centrifuge at 8000g for 10 min at 4°C. Collect the supernatant and keep it on ice for testing.
Liquid samples: Test directly.
2. Assay Procedure
- Preheat the microplate reader for 30 min. Set the wavelength to 412 nm and zero with distilled water.
- Prepare the working solution before use by adding 18 mL Reagent I to the Reagent II bottle. Mix thoroughly and set aside. Aliquot unused reagent and store at -20°C. Repeated freezing and thawing is prohibited.
- Prepare Reagent III immediately before use by adding 1 mL anhydrous ethanol. Dissolve completely and set aside. Store unused reagent at 4°C.
- Blank well: add 10 μL extract and 180 μL working solution to a 96-well plate. Mix thoroughly and react at 35°C for 15 min. Add 10 μL Reagent III, mix thoroughly, and let stand at 25°C for 10 min. Measure absorbance at 412 nm and record as Ablank.
- Assay well: add 10 μL sample and 180 μL working solution to a 96-well plate. Mix thoroughly and react at 35°C for 15 min. Add 10 μL Reagent III, mix thoroughly, and let stand at 25°C for 10 min. Measure absorbance at 412 nm and record as Aassay.
Calculate ΔA as follows: ΔA = Aassay- Ablank. Only one blank well is required.
3. Calculation
Use the following formulas for the 96-well plate assay.
Calculation According to Protein Concentration
Definition of activity unit: the amount of enzyme that catalyzes the production of 1 nmol TNB per milligram of protein per minute is defined as one enzyme activity unit.
AAT (nmol/min/mg prot) = ΔA ÷ (ε × d) × Vtotal reaction÷ (Vsample× Cpr) ÷ T = 196.08 × ΔA ÷ Cpr
Calculation According to Sample Mass
Definition of activity unit: the amount of enzyme that catalyzes the production of 1 nmol TNB per gram of tissue per minute is defined as one enzyme activity unit.
AAT (nmol/min/g fresh weight) = ΔA ÷ (ε × d) × Vtotal reaction÷ (W × Vsample÷ Vtotal sample) ÷ T = 196.08 × ΔA ÷ W
Calculation According to Cell Number
Definition of activity unit: the amount of enzyme that catalyzes the production of 1 nmol TNB per 104cells per minute is defined as one enzyme activity unit.
AAT (nmol/min/104cells) = ΔA ÷ (ε × d) × Vtotal reaction÷ (cell number × Vsample÷ Vtotal sample) ÷ T = 196.08 × ΔA ÷ cell count
Calculation According to Liquid Volume
Definition of activity unit: the amount of enzyme that catalyzes the production of 1 nmol TNB per milliliter of sample per minute is defined as one enzyme activity unit.
AAT (nmol/min/mL) = ΔA ÷ (ε × d) × Vtotal reaction÷ Vsample÷ T = 196.08 × ΔA
Formula Parameters
| ε | TNB extinction coefficient: 13600 L/mol/cm |
|---|---|
| d | 96-well plate optical path length: 0.5 cm |
| Vtotal reaction | Total reaction volume: 0.2 mL |
| Vsample | Volume of supernatant added to the reaction system: 0.01 mL |
| Vtotal sample | Volume of Extraction Solution added: 1 mL |
| Cpr | Protein content: mg/mL |
| W | Sample mass: g |
| T | Reaction time: 15 min |
Precautions
- Before the formal assay, select 2-3 samples with large expected differences for a preliminary test.
- Required instruments and supplies not provided: mortar, ice, benchtop centrifuge, microplate reader, 96-well plate, constant-temperature water bath, and absolute ethanol.
- This 100T kit tests 96 samples.
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