Ferric-Chelate Reductase (FCR) Activity Assay Kit - Microplate Method

Product Code: F930372

Product Introduction

Ferric-chelate reductase (FCR) catalyzes the reduction of ferric chelates from Fe³⁺to Fe²⁺and plays an important role in iron uptake in some species.

In this assay, FCR reduces Fe³⁺to Fe²⁺. The Fe²⁺then reacts with ferrozine to produce color with a characteristic absorbance at 562 nm.

Package Contents

Quality Standards and Safety

Transportation and Storage

Transportation: Transport with ice packs.

Storage: Store at 2-8°C, protected from light. Shelf life: 180 days.

Required Instruments and Supplies

Microplate reader, benchtop centrifuge, adjustable pipette, 96-well plate, mortar, ice, and distilled water.

Instructions for Use

1. Preparation of Crude Enzyme Extract

1. Prepare the extract using a tissue mass (g) to water (mL) ratio of 1:5-10.
2. It is recommended to weigh about 0.1 g of tissue and add 1 mL of distilled water.
3. Homogenize in an ice bath.
4. Centrifuge at 10000 g, 4°C for 10 min.
5. Collect the supernatant and keep it on ice for testing.

2. Assay Procedure

1. Preheat the microplate reader for more than 30 min, set the wavelength to 562 nm, and zero with distilled water.
2. Prepare the working solution by mixing Reagents I, II, and III at a 1:1:1 ratio. Prepare immediately before use and only in the amount needed.
3. In a 96-well plate, add 50 μL of sample supernatant and 150 μL of working solution.
4. Mix well and record the initial absorbance value A1.
5. Record the absorbance again after 30 min as A2.
6. Calculate ΔA = A2 - A1.

Activity Calculation

Using a 96-well plate, the standard curve is:

y = 4.0007x + 0.0011, R²= 0.9997

y: absorbance.

3.1 Calculated by Sample Mass

Unit definition: the amount of enzyme that produces 1 nmol of Fe²⁺-ferrozine per minute per gram of sample is defined as one enzyme activity unit.

FCR (nmol/min/g fresh weight) = (ΔA - 0.0011) ÷ 4.0007 × 1000 × V_std÷ (V_sample÷ V_{total sample}× W) ÷ T = 8.331 × (ΔA - 0.0011) ÷ W

3.2 Calculated by Sample Protein Concentration

Unit definition: the amount of enzyme that produces 1 nmol Fe²⁺-ferrozine per minute per mg protein is defined as one enzyme activity unit.

FCR (nmol/min/mg prot) = (ΔA - 0.0011) ÷ 4.0007 × 1000 × V_std÷ (V_sample÷ V_{total sample}× Cpr) ÷ T = 8.331 × (ΔA - 0.0011) ÷ Cpr

Parameter Definitions

Precautions

Before the formal assay, select 2-3 samples with large expected differences for pretesting.