Ornithine Aminotransferase (δ-OAT) Activity Assay Kit, Micro Method

Product Information

Product Introduction

Proline is an important osmotic regulator in plants and supports adaptation to stress conditions. In higher plants, proline metabolism includes glutamate (Glu) and ornithine (Orn) synthesis pathways, classified by their initial substrates.

Ornithine aminotransferase (δ-OAT) is a key enzyme in the pathway that uses ornithine as a precursor for proline synthesis. Ornithine and α-ketoglutaric acid undergo an aminotransferase reaction catalyzed by δ-OAT in the presence of NADH, generating pyrroline-5-carboxylic acid (P5C) and NAD. The change in absorbance at 340 nm reflects δ-OAT activity.

Package Contents

Quality and Safety Information

Transportation and Storage

Instructions for Use

1. Enzyme Solution Extraction

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 tissue and add 1 mL extraction solution.

1. Add the extraction solution to the tissue sample.
2. Homogenize in an ice bath.
3. Centrifuge at 4°C and 10000g for 10 min.
4. Keep the supernatant on ice for testing.

Cell Samples

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 500 x 10,000 cells.

1. Disrupt the cells by ultrasonication in an ice bath at 300 W, with 3 seconds ultrasound and 7 seconds interval, for a total time of 3 min.
2. Centrifuge at 4°C and 10000g for 10 min.
3. Keep the supernatant on ice for testing.

Liquid Samples

Test liquid samples directly.

2. Reagent Preparation

1. Before use, add 8 mL Reagent I to Reagent II and dissolve thoroughly. Store unused prepared reagent at 4°C.
2. Before use, add 8 mL Reagent I to Reagent III and dissolve thoroughly. Store unused prepared reagent at 4°C.
3. Before use, add 4 mL Reagent I to each bottle of Reagent IV and dissolve thoroughly. Prepare fresh before use.

Powdered reagents must be prepared by the user before the assay.

3. Assay Procedure

1. Preheat the microplate reader for 30 min and set the wavelength to 340 nm.
2. Preheat the prepared Reagents II, III, and IV at 37°C for 5 min.
3. In a 96-well plate, add 60 μL Reagent II, 60 μL Reagent III, 60 μL Reagent IV, and 20 μL crude enzyme solution in sequence.
4. Mix thoroughly.
5. Record the initial absorbance at 340 nm as A1.
6. React at 37°C for 10 min, then record the absorbance as A2.
7. Calculate ΔA = A1 - A2.

Activity Calculation

Calculation by Sample Protein Concentration

Unit definition: the consumption of 1 nmol 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) × V_{total reaction}÷ (V_sample× Cpr) ÷ T = 321.54 × ΔA ÷ Cpr

Calculation by Sample Mass

Unit definition: the consumption of 1 nmol NADH per gram of tissue per minute is defined as one unit of enzyme activity.

δ-OAT (nmol/min/g fresh weight) = ΔA ÷ (ε × d) × V_{total reaction}÷ (W × V_sample÷ V_{sample total}) ÷ T = 321.54 × ΔA ÷ W

Calculation by Cell Number

Unit definition: the consumption of 1 nmol NADH per 10⁴cells per minute is defined as one unit of enzyme activity.

δ-OAT (nmol/min/10⁴cells) = ΔA ÷ (ε × d) × V_{total reaction}÷ (V_sample× cell number ÷ V_{sample total}) ÷ T = 321.54 × ΔA ÷ cell count

Calculation by Liquid Volume

Unit definition: the consumption of 1 nmol NADH per milliliter of liquid per minute is defined as one unit of enzyme activity.

δ-OAT (nmol/min/mL) = ΔA ÷ (ε × d) × V_{reaction total}÷ V_sample÷ T = 321.54 × ΔA

Formula Parameters

Precautions

• Before the formal assay, select 2-3 samples with large expected differences for preliminary testing.
• Required instruments and supplies include a balance, refrigerated centrifuge, mortar, microplate reader, and 96-well plate.