112907 Cis-Aconitase Activity Assay Kit - Spectrophotometric Method

Product Introduction

Aconitase catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid cycle. Citrate is not readily oxidized. Through dehydration and hydration reactions, aconitase shifts the hydroxyl group from the β-carbon atom to the α-carbon atom to form isocitrate, which is readily dehydrogenated and oxidized for the subsequent oxidative decarboxylation reaction.

ACO catalyzes the conversion of citrate to isocitrate, and the oxidative decarboxylation of isocitrate reduces NAD⁺to generate NADH, increasing light absorption at 340 nm.

Sample detection results may vary depending on the detection conditions and instrument. Any figure data are for reference only.

Package Contents

Quality Standards and Safety

Transportation and Storage

Transportation: Transport with ice packs.

Storage: Store Reagent IV, Reagent V, and Reagent VII at 2-8 °C. Store the remaining components at -20 °C. Shelf life: 180 days.

Instructions for Use

1. Sample Processing

1. Weigh about 0.1 g tissue or collect 5 million cells. Add 1 mL Reagent I and 10 μL Reagent III, then homogenize using an ice-bath homogenizer or mortar.
2. Transfer the homogenate to a centrifuge tube and centrifuge at 600 g, 4 °C for 5 min.
3. Discard the pellet. Transfer the supernatant to another centrifuge tube and centrifuge at 11000 g, 4 °C for 10 min.
4. The supernatant is the cytoplasmic extract and can be used to determine cytoplasmic cis-aconitase activity.
5. Add 200 μL Reagent II and 2 μL Reagent III to the pellet from step 4. Disrupt by sonication in an ice bath at 20% power or 200 W, sonicate for 3 s at 10 s intervals, and repeat 30 times. Use this for determination of mitochondrial cis-aconitase activity.

2. Reagent Preparation

1. Add 3 mL distilled water to Reagent VI and dissolve thoroughly before use. Prepare fresh and use immediately.
2. Add 36 mL Reagent IV to Reagent VII and dissolve thoroughly before use.
3. Working solution: before use, add 36 mL of Reagent VII, then add 3 mL distilled water, 3 mL Reagent IV, 3 mL Reagent V, and Reagent VI. Mix thoroughly. Prepare fresh and use immediately, or aliquot the working solution and store at -20 °C for up to one week.

3. Assay Procedure

1. Preheat the spectrophotometer for more than 30 min and set the wavelength to 340 nm. Use distilled water to zero the instrument.
2. Place the working solution in a 37 °C water bath for mammals or a 25 °C water bath for other species for 10 min.
3. In a 1 mL quartz cuvette, add 100 μL sample and 900 μL working solution. Mix well. Immediately record the absorbance at 340 nm at 20 s as A1 and at 3 min 20 s as A2. Calculate ΔA = A2 - A1.

Activity Calculation

1. Based on Protein Concentration

Unit definition: one unit is the amount of enzyme that generates 1 nmol of NADH per minute per mg tissue protein.

ACO activity (nmol/min/mg prot) = [ΔA × V_{total reaction}÷ (ε × d) × 10⁹] ÷ (V_sample× Cpr) ÷ T = 536 × ΔA ÷ Cpr

2. Based on Fresh Weight

Unit definition: one unit is the amount of enzyme that generates 1 nmol of NADH per minute per g tissue.

ACO (nmol/min/g fresh weight) = [ΔA × V_{total reaction}÷ (ε × d) × 10⁹] ÷ (W × V_sample÷ V_{total sample}) ÷ T = 108 × ΔA ÷ W

3. Based on Bacterial or Cell Density

Unit definition: one unit is the amount of enzyme that generates 1 nmol of NADH per minute for every 1 × 10⁴bacteria or cells.

ACO activity (nmol/min/10⁴cells) = [ΔA × V_{total reaction}÷ (ε × d) × 10⁹] ÷ (500 × V_sample÷ V_{total sample}) ÷ T = 0.722 × ΔA

Precautions

1. This 50T kit can test 48 samples.
2. Required instruments and supplies: UV spectrophotometer, water bath, benchtop centrifuge, adjustable pipette, 1 mL quartz cuvette, mortar, ice, and distilled water.
3. Before formal measurement, perform a preliminary test using 2-3 samples with relatively large expected differences.