Iron Content Assay Kit (Ferrozine, Micro Method) - BioReagent

Cat. No.: I1505775

GRADE & PURITY BioReagent

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Application

Cell Metabolism

Overview

  Iron is an essential trace element in the human body, with a total content of approximately 3270 mg. It is widely distributed: 67.6% of iron is found in hemoglobin as a prosthetic group, involved in oxygen transport; 2.59% and 4.15% are present in bone and myoglobin, respectively; and storage iron accounts for about 25.37%. Serum iron is bound to transferrin in the form of ferric ions (Fe³⁺). Therefore, measuring serum iron requires first dissociating Fe³⁺ from transferrin.

Assay Principle

  This assay utilizes a microplate method with Ferrozine as the chromogen. In an acidic medium, serum iron bound to transferrin is released. Iron in other samples is also liberated under acidic conditions. The released iron is then reduced to ferrous ions (Fe²⁺) by a reducing agent (present in the assay buffer). The Fe²⁺ subsequently reacts with Ferrozine to form a magenta-colored complex. The absorbance at 562 nm is measured using a microplate reader. This method is suitable for detecting iron content in samples such as serum, plasma, and tissues. As this direct detection method is used, a serum blank should be included to correct for the sample's inherent color. The iron content is calculated according to the formula. This kit shows a good linear relationship up to 140 μmol/L. Triglycerides ≤ 3.39 mmol/L and bilirubin ≤ 171 μmol/L basically do not interfere with this method. This kit is for research use only and not suitable for clinical diagnosis or other purposes.

I1505775	Component	100T	Storage
I1505775A	Iron Standard (100 μg/mL)	1 mL	2-8℃. Store in the dark.
I1505775B	Iron Standard Diluent	2 mL	RT
I1505775C	Fe Assay Buffer	25 mL	2-8℃
I1505775D	Ferrozine Chromogenic Solution	1 mL	2-8℃. Store in the dark.
I1505775E	ddH₂O	10 mL	RT

Required Materials Not Provided

  Centrifuge tubes or test tubes, Microplate reader, 96-well plate

Experimental Procedure

1. (Optional) Sample Preparation

1.1 Plasma/Serum Samples

• Prepare plasma or serum by conventional methods.

• Store at -20°C for Fe detection.

1.2 Cell or Tissue Samples

• Homogenize an appropriate amount of cells or tissue.

• Centrifuge at low speed and collect the supernatant.

• Store at -20°C for Fe detection.

1.3 High-Concentration Samples

• If the sample contains a high concentration of Fe, dilute it with ddH₂O. Do not use regular distilled water for dilution.

Note: (Optional) After preparation, determine the protein concentration using a BCA Protein Assay Kit to calculate Fe content per mg protein.

2. Preparation of Iron Standard Working Solution

• Dilute the Iron Standard (100 μg/mL) with Iron Standard Diluent at a 1:49 ratio to prepare the Iron Standard Working Solution (2 μg/mL).

• Store at 4°C protected from light; stable for 3 months.

3. Fe Assay Setup

• Use a 96-well plate cleaned with dilute hydrochloric acid and rinsed with deionized water.

• Set up Blank, Standard, and Test wells according to the table below.

• Add reagents in the specified order, avoiding bubbles.

• If the sample's iron concentration is too high, reduce the sample volume or dilute appropriately before assaying. Duplicate wells are recommended.

Reagent (μL)	Blank Well	Standard Well	Test Well
ddH₂O	75	—	—
Iron Standard Working Solution (2 μg/mL)	—	75	—
Sample	—	—	75
Fe Assay Buffer	200	200	200

Mix well. Read absorbance at 562nm, zeroed with the Blank well. Record for Test well (This is the Serum Blank, 

Ferrozine Chromogenic Solution

8.5

8.5

8.5

4. Fe Measurement

• Mix well.

• Incubate at room temperature for 15 minutes or at 37°C for 10 minutes.

• Zero the instrument with the Blank well.

• Measure the absorbance of the Standard and Test wells at 562 nm (recorded as A_standard and A_test).

• Complete the colorimetric measurement within 1 hour.

5. Calculation

5.1 Plasma Iron, Serum Iron

Fe (μmol/L) = { [A_test - (A_{serum blank} × 0.97)] / A_standard } × 35.8 Fe (mg/L) = { [A_test - (A_{serum blank} × 0.97)] / A_standard } × 2 

5.2 Tissue Iron, Cell Iron 

Fe (μmol/g prot) = { [A_test - (A_{serum blank} × 0.97)] / A_standard } × 35.8 / Pr 

Fe (mg/g prot) = { [A_test - (A_{serum blank} × 0.97)] / A_standard } × 2 / Pr 

Fe (μmol/N cells) = { [A_test - (A_{serum blank} × 0.97)] / A_standard } × 35.8 / (N × V_sample / V_{total extract}) 

Fe (mg/N cells) = { [A_test - (A_{serum blank} × 0.97)] / A_standard } × 2 / (N × V_sample / V_{total extract}) 

Parameter Definitions: 

A_test: Absorbance of the Test well after adding Ferrozine Chromogenic Solution. 

A_{serum blank}: Absorbance of the Test well before adding Ferrozine Chromogenic Solution. 

A_standard: Absorbance of the Standard well. 

Pr: Protein concentration of the test sample (g/L). 

N: Total number of cells or bacteria. 

V_sample: Volume of sample added to the reaction system (L). 

V_{total extract}: Total volume of the sample extraction buffer (L).

Unit Conversions:

• Iron Standard (2 μg/mL) = Iron Standard (2 mg/L) = Iron Standard (35.8 μmol/L)

• μg/dL = μmol/L / 0.179

• μmol/L = 0.179 × μg/dL = 17.9 × μg/mL

Reference Interval:

• Healthy Adults Serum Iron:

  • Male: 11 ~ 30 μmol/L (60 ~ 170 μg/dL)

  • Female: 9 ~ 27 μmol/L (50 ~ 150 μg/dL)

6. Standard Curve Reference

*(Placeholder for standard curve graph and data, typically showing absorbance vs. concentration for standards like 0, 0.5, 1, 2, 4, 6, 8, 10 μg/mL, measured at 562 nm)*

*Note: Standard curves may vary due to differences in instruments and operational techniques. Empirical data suggests deviations in the standard curve at iron standard concentrations below 0.1 μg/mL and above 40 μg/mL.*

Precautions

1. Hemolyzed samples interfere with the assay; avoid using them if possible.

2. If the sample concentration is too high, dilute with distilled water and re-assay, multiplying the result by the dilution factor.

3. Use high-purity deionized water during the experiment; do not use ordinary distilled water.

4. Glassware should be soaked in 10% hydrochloric acid for 24 hours and rinsed with deionized water before use.

5. Avoid contact with iron objects to prevent iron contamination.

6. The standard color is stable for 24 hours. The serum color is stable for 30 minutes; the color intensifies slowly over time, so measurement must be completed within 1 hour.

7. The value 0.97 is a volume correction factor.

8. The within-run coefficient of variation (CV) for this method is ≤ 3.1%; the between-run CV is ≤ 2.6%.

9. Use reagents promptly after opening to avoid affecting subsequent experimental results.

10. For your safety and health, wear lab coats and disposable gloves during operation.

Specifications

Specifications & Purity

BioReagent

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Storage

Store at 2-8°C, Protected from light, Room temperature

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive