Application

This kit can be used to measure the concentration of iron in tissue samples. 

Detection significance

Iron is an essential element in most life forms, from bacteria to mammals. It is commonly found in metalloprotein centers, heme complexes and oxygen carrier proteins. The main form of iron stored in human body is ferritin. Iron is essential for a variety of metabolic processes, including oxygen transport, DNA synthesis and electronic transport.

Detection principle

Under the action of acidic solution and reductant, Fe3+ can be reduced into Fe2+. The latter then bind to bipyridine and form pink complexes. The color develops in proportion to the amount of iron ions within a certain range. The concentration of iron can be calculated by measuring the OD value at 520 nm.

Experimental instruments

Tubes, Micropipet, Vortex Mixer, Centrifuge, Water bath, Spectrophotometer (520 nm）

Preparation of sample

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

10% Tissue homogenate: Weigh 0.02-1.0 g tissue, rinse with distilled water and dry with filter paper, then weigh the tissue. Add PBS (0.01 M，pH 7.4) according to the ratio of Weight (g): Volume (mL)=1: 9, then make the mechanical homogenization in ice water bath to prepare 10% homogenate. Centrifuge  at 10000 g for10 min. Take the supernatant and preserve it on ice for detection.

Operation steps

1. Blank tube: Add 0.5 mL of Double-distilled water into a 5 mL centrifuge tube.

Standard tube: Add 0.5 mL of 2 mg/mL Iron Standard working solution into a 5 mL centrifuge tube.

Sample tube: Add 0.5 mL of Sample into a 5 mL centrifuge tube.

2. Add 1.5 mL of Iron Chromogenic agent, mix fully with vortex mixer, then incubate in 100℃ water bath for 5 min. (Blank tube and standard tube can be treated without 100℃ water bath.)

3. Cool the tubes with running water, centrifuge the tubes at 2300 g for 10 min. (If the supernatant is still turbid, take the turbid supernatant into another centrifuge tube and centrifuge again.)

4. Take 1.0 mL supernatant. Set spectrophotometer to zero with double-distilled water, and measure the OD value of each tube at 520 nm with 0.5 cm optical path diameter cuvette.

Note: It can be refer to the following operating table

[Note]: When taking the supernatant for colorimetry measurement, it is suggested to take the supernatant carefully with the pipette to avoid adding sediment to cuvette and affect the OD value.

Technical parameters

1. The sensitivity of the kit is 0.072 mg/L.

2. The intra-assay CV is 2.5 % and the inter-assay CV is 4.6 %.

3. The recovery of the kit is 99 %.

4. The linear range of the kit is 0.072-60 mg/L.

Notes

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3.The validity of kit is 6 months.

4. Do not use components from different batches of kit.