Ornithine Aminotransferase(δ-OAT) Activity Assay Kit
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Ornithine Aminotransferase Activity Assay Kit - Microplate Method
Product code: 55794
Assay target: Ornithine Aminotransferase (δ-OAT) Activity
Product Introduction
Proline is an important osmotic regulator that helps plants adapt to stress conditions. In higher plants, proline metabolism is divided into two synthesis pathways according to the initial substrate: the glutamate (Glu) pathway and the ornithine (Orn) pathway.
Ornithine aminotransferase (δ-OAT) is a key enzyme in the pathway that synthesizes proline using ornithine as the precursor, and it plays an important role in plant adaptation to stress conditions.
Under the action of ornithine aminotransferase and NADH, ornithine and α-ketoglutaric acid undergo an aminotransferase reaction to form pyrroline-5-carboxylic acid (P5C), while producing NAD. The activity level of ornithine aminotransferase is reflected by measuring the change in absorbance at 340 nm.
Package Contents
| Size | Code | Component | Quantity |
|---|---|---|---|
| 100T | 55794.1 | Reagent I | 1 bottle |
| 100T | 55794.2 | Reagent II | 1 bottle |
| 100T | 55794.3 | Reagent III | 1 bottle |
| 100T | 55794.4 | Reagent IV | 1 bottle |
| 100T | 55794.5 | Extraction Solution | 1 bottle |
| 100T | 55794.m | Instructions | 1 copy |
Quality Standards and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | -- | -- |
| Reagent II | -- | -- |
| Reagent III | -- | -- |
| Reagent IV | -- | -- |
| Extraction Solution | -- | -- |
Transportation and Storage
| Transportation | Transport with ice packs. |
|---|---|
| Storage | Store Reagent IV at -20°C. Store all other components at 2-8°C protected from light. Shelf life is 180 days. |
Instructions for Use
1. Enzyme Solution Extraction
1.1 Tissue Samples
Use a sample mass (g) to extraction solution volume (mL) ratio of 1:5-10. It is recommended to weigh approximately 0.1 g of sample and add 1 mL of Extraction Solution.
- Add the Extraction Solution to the tissue sample.
- Homogenize in an ice bath.
- Centrifuge at 4°C and 10000g for 10 min.
- Collect the supernatant and keep it on ice for testing.
1.2 Cell Samples
Use a cell number (104cells) to extraction solution volume (mL) ratio of 500-1000:1. It is recommended to add 1 mL of Extraction Solution to 500 ten thousand cells.
- Disrupt the cells by ultrasonic treatment in an ice bath at 300 W: ultrasonication for 3 seconds, interval for 7 seconds, total time 3 min.
- Centrifuge at 4°C and 10000g for 10 min.
- Collect the supernatant and keep it on ice for testing.
1.3 Liquid Samples
Test liquid samples directly.
2. Reagent Preparation
- Before use, add 8 mL of Reagent I to Reagent II and dissolve thoroughly. Store unused prepared reagent at 4°C.
- Before use, add 8 mL of Reagent I to Reagent III and dissolve thoroughly. Store unused prepared reagent at 4°C.
- Before use, add 4 mL of Reagent I to each bottle of Reagent IV and dissolve thoroughly. Prepare fresh and use immediately.
3. Assay Procedure
- Preheat the microplate reader for 30 min and set the wavelength to 340 nm.
- Preheat the prepared Reagents II, III, and IV at 37°C for 5 min.
- In a 96-well plate, sequentially add 60 µL of Reagent II, 60 µL of Reagent III, 60 µL of Reagent IV, and 20 µL of crude enzyme solution.
- Mix thoroughly.
- Record the initial absorbance at 340 nm as A1.
- React at 37°C for 10 min, then record the absorbance at 340 nm as A2.
- Calculate ΔA = A1 - A2.
Powdered reagents need to be prepared by the user.
Activity Calculation
1. Calculation by Sample Protein Concentration
Definition of enzyme activity unit: consumption of 1 nmol of NADH per minute per milligram of tissue protein is defined as one unit of enzyme activity.
δ-OAT (nmol/min/mg prot) = ΔA ÷ (ε × d) × Vtotal reaction÷ (Vsample× Cpr) ÷ T = 321.54 × ΔA ÷ Cpr
2. Calculation by Sample Mass
Definition of enzyme activity unit: consumption of 1 nmol of NADH is defined as one enzyme activity unit.
δ-OAT (nmol/min/g fresh weight) = ΔA ÷ (ε × d) × Vtotal reaction÷ (W × Vsample÷ Vtotal sample) ÷ T = 321.54 × ΔA ÷ W
3. Calculation by Cell Number
Definition of enzyme activity unit: consumption of 1 nmol of NADH per minute per 104cells is defined as one enzyme activity unit.
δ-OAT (nmol/min/104cells) = ΔA ÷ (ε × d) × Vtotal reaction÷ (Vsample× number of cells ÷ Vtotal sample) ÷ T = 321.54 × ΔA ÷ number of cells
4. Calculation by Liquid Volume
Definition of enzyme activity unit: consumption of 1 nmol of NADH per milliliter of liquid per minute is defined as one enzyme activity unit.
δ-OAT (nmol/min/mL) = ΔA ÷ (ε × d) × Vtotal reaction÷ Vsample÷ T = 321.54 × ΔA
Formula Parameters
| Vtotal reaction | Total volume of the reaction system, 0.2 mL |
|---|---|
| ε | NADH molar extinction coefficient, 6.22 × 103L/mol/cm |
| d | 96-well plate optical path length, 0.5 cm |
| Vsample | Added sample volume, 0.02 mL |
| Vtotal sample | Added extraction solution volume, 1 mL |
| T | Reaction time, 10 min |
| Cpr | Sample protein concentration, mg/mL |
| W | Sample mass, g |
Precautions
- Before formal measurement, select 2-3 samples with large expected differences for preliminary testing.
- Instruments and supplies required but not provided: balance, refrigerated centrifuge, mortar, microplate reader, and 96-well plate.
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