Iron Assay Kit ab83366 provides a simple convenient means of measuring ferrous (Fe2+) and/or ferric (Fe3+) iron in biological samples.
Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.
Iron Assay Kit ab83366 provides a simple convenient means of measuring ferrous (Fe2+) and/or ferric (Fe3+) iron in biological samples.
Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.
Iron Assay Kit ab83366 provides a simple convenient means of measuring ferrous (Fe2+) and/or ferric (Fe3+) iron in biological samples.
How the assay works
This ferrozine assay kit / iron assay kit uses an improved variant of the ferrozine molecule (ferene) which has been shown to give more accurate results for biological iron levels; that are closer to the results seen with mass spectrometry.
In a ferrozine assay, free ferrous iron (Fe2+) reacts with the ferrozine molecule (in this case the ferene molecule) to produce a stable colored complex with absorbance that can be measured with a colorimetric plate reader. The ferene iron probe used in this assay kit has absorbance at 593 nm when complexed with ferrous iron (Fe2+).
In Iron Assay Kit ab83366, in addition to the ferene iron probe and iron standard, an acidic assay buffer, with pH less than 5.5, is provided to release iron from iron carrier proteins. A reducing agent is also provided to convert ferric iron (Fe3+) to form Fe2+.
Ferrous iron (Fe2+) can be measured alone, or by addition of the reducing agent the level of total iron (Fe2+ and Fe3+) can be measured. The level of ferric iron (Fe3+) is calculated by subtracting ferrous iron from total iron.
A specific chelate chemical is included in the buffer to block copper ion (Cu2+) interference.
The kit measures iron in the linear range of 0.4 to 10 nmol or 8 μM to 200 μM.
Iron assay protocol summary
Getting the best performance from ab83366
This assay is not suitable for use with samples containing EDTA or citrate.
Related and recommended products
Iron Assay Kit ab83366 is often used in combination with Lipid Peroxidation (MDA) Assay Kit Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970 and GSH/ GSSG Assay Kit GSH+GSSG / GSH Assay Kit (Colorimetric) ab239709 in the study of ferroptosis.
To measure total iron binding capacity and iron levels in serum, we recommend Total Iron-Binding Capacity (TIBC) and Serum Iron Assay Kit Total Iron-Binding Capacity (TIBC) and Serum Iron Assay Kit ab239715.
For iron staining in tissues, we recommend Iron Stain Kit (Prussian Blue Stain) Iron Stain Kit (Prussian Blue Stain) ab150674.
Other notes
This product is manufactured by BioVision, an Abcam company and was previously called K390 Iron Colorimetric Assay Kit. K390-100 is the same size as the 100 test size of ab83366.
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
Iron is a chemical element essential for various metabolic processes. It functions as a cofactor for proteins involved in oxygen transport and electron transfer. Iron exists in several forms including ferritin and hemosiderin and has a molecular mass of approximately 55.85 dalton. In human bodies iron is expressed mainly in red blood cells liver muscle tissues and is stored in ferritin complexes. For histological studies techniques such as iron staining and Perls Prussian blue are used to visualize iron in tissues. Additionally assays of iron and iron TIBC (Total Iron Binding Capacity) measure iron levels and binding affinity.
Iron plays a role in cellular respiration DNA synthesis and electron transport. It is essential in hemoglobin within red blood cells and myoglobin in muscles where it binds and transports oxygen. Iron is also part of the iron-sulfur cluster and heme complexes important for electron exchange in cells. The blue stain used in histology highlights areas rich in iron aiding in diagnosis and research. You can assess iron availability and transport through tests like iron + TIBC.
Iron is integral to oxygen transport and mitochondrial electron transport chain regulation. It is important in erythropoiesis and energy production. Iron works with proteins like transferrin and ferritin important for maintaining iron homeostasis. Cytochrome oxidase a heme-containing enzyme plays an essential role in electron transport linked with iron. Iron is also vital in modulating cellular oxidative stress pathways which help in managing reactive oxygen species.
Abnormal iron metabolism leads to conditions like anemia and hemochromatosis. In anemia deficiency of iron results in reduced oxygen transport due to low hemoglobin levels. Hemochromatosis on the other hand involves iron overload damaging organs like the liver and heart. The balance of iron is regulated by proteins including transferrin and ferritin which maintain necessary levels for cellular function and prevent toxic accumulation. The use of iron staining in histology assists in identifying iron-related pathologies.
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Iron measured in human urine showing concentration (micromolar)
Leal SM Jr et al examined if iron availability regulates fungal growth in an infection as fungal infection intiates an iron sequestration response. Mice given Fe-dextran (Fe-Dex) and deferoxamine (Defox) shows a higher fungal mass (Fungal dsRed) compared to vehicle treated mice over 48 hours. Iron content was quantified in mouse serum using Iron assay kit (ab83366).
Assay of soluble free iron from a soil sample (5 μL of 100 μL buffer into which 100 mg of soil had been stirred), 5 μL of FBS and 5 μL of a 100 μM sample of iron standard.
Example of iron standard curve using ab83366.
Assay of total iron(II + III) in off-the-clot human serum (50 μl of serum was added per well). Data are mean ± SEM from 2 independent replicates, performed in duplicate wells.
Tang et al. used Iron Assay Kit ab83366 and MDA assay kit Lipid Peroxidation (MDA) Assay Kit (Colorimetric) ab233471 to investigate a ferroptosis resistance mechanism involving the upregulation of TXNDC12, a thioredoxin domain-containing protein located in the endoplasmic reticulum.
Iron assays indicated that erastin- or RSL3-induced iron accumulation in HL60 and K562 cells during ferroptosis was unaffected by the knockdown of TXNDC12. In contrast, the absence of TXNDC12 accelerated erastin- or RSL3-induced lipid peroxidation, as evidenced by the malondialdehyde (MDA) assay.
The concentration of MDA in cells or tissues was determined using an MDA assay kit (Abcam, Lipid Peroxidation (MDA) Assay Kit (Colorimetric) ab233471) following the manufacturer's instructions. The kit provided MDA standard and all necessary buffers. The assay involved the following main steps: serial dilution of MDA standards and test samples; addition of 10 µl of MDA color reagent stock solution to each well of MDA standard; incubation at room temperature for 10-30 minutes; addition of 40 µl of reaction solution and further incubation at room temperature for 30-60 minutes; measurement of the end-point absorbance at OD 695-700 nm using an absorbance plate reader with path-check correction.
The relative level of free ferrous iron (Fe2+) in cells was determined using an iron assay kit (Abcam, ab83366) following the manufacturer's instructions. The kit provided the iron standard and all necessary buffers. The assay involved the following main steps: setting up reaction wells; adding 5 µL of iron reducer to each standard well; adding 5 µL of assay buffer to each sample; mixing and incubating the standards and samples at 37°C for 30 minutes; adding 100 µL of iron probe to each well containing the iron standard and test samples; mixing and incubating them at 37°C for 60 minutes, protected from light. The output was measured immediately using a colorimetric microplate reader at OD 593 nm.
Diagram showing the principles of the Iron assay method.
Junhing et al. used Iron Assay Kit ab83366 with a model of myocardial ischemia/reperfusion injury in rats treated with diltiazem and (-) epicatechin.
The concentration of Fe2+ in rat cardiac tissues: Control; I/R; L-EPI (I/R + L-EPI: 1 mg/kg/day of EPI); H-EPI (I/R + H-EPI: 2 mg/kg/day of EPI), DIL (I/R + DIL).
The total iron levels were measured in heart tissue in each group using the Iron Assay kit (ab83366, Abcam, UK). Tissues homogenate was lysed in 4 volume of buffer and centrifuged at 16,000 Ч g for 10 min. 5 µL of iron reducer agent was added into 50 µL of samples for Fe2+ assay, and 100 µL of iron probe solution was added to the samples and incubated at 25° C for 60 min kept in dark place. The absorbance of samples at 593 nm was measured using a micro spectrophotometer (Nanodrop, Thermo Fisher Scientific, USA).
Wang et al. used Iron Assay Kit ab83366 and Lipid Peroxidation Assay Kit Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970 to investigate ferroptosis in a mouse model of intestinal ischemia-reperfusion.
Ferroptosis occurs in a mouse model of intestinal I/R. Total iron, Fe2+, Fe3+, MDA, total glutathione, GSH, GSSG, and GSH/GSSG levels were evaluated in reperfusion tissues (n = 8).
To simulate intestinal ischemia-reperfusion (I/R), mice were anesthetized with 5% isoflurane and disinfected with Aner iodine. A midline incision was made in the upper abdomen to expose and dissociate the mesenteric artery. Closure of the mesenteric artery was performed for 60 min with a small artery clip to simulate the ischemic process. The arterial clamp was released to simulate the reperfusion process. Three different reperfusion times, namely, 15 min, 30 min, and 60 min, were used.
The total iron, Fe2+ and Fe3+ levels were assessed with an iron test kit (ab83366, Abcam) with a microplate reader at OD 593 nm. Malondialdehyde (MDA) was assessed with a Lipid Peroxidation Assay kit (Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970, Abcam) at OD 532 nm. Tissue samples and cell samples were prepared according to the manufacturer’s protocols.
Deng et al. used Iron Assay Kit ab83366 to investigate iron levels in fatty liver.
Concentration of liver tissue Fe2+ and liver tissue Fe3+ between NC (normal diet) group and HFD (high fat diet) group (n?=?5).
The iron concentration of liver tissue was detected by an Iron Assay Kit (No. ab83366, Abcam, UK), according to the instructions provided by the manufacturer. Briefly, a standard dilution was prepared in the standard wells (100 ?l), and the sample wells (40 ?l) were prepared with sample solution (the volume was adjusted to 100 ?l/well using iron assay buffer). Then, 5 ?l of iron reducer was added to each standard well. The concentration of Fe2+ in the liver tissue samples was determined by adding 5 ?l of assay buffer to each sample. To determine the total iron (Fe2+?+?Fe3+) concentration of the liver tissue samples, 5 ?l of iron reducer was added to each sample. Then, the reagents were mixed and incubated for 30 min in an oven at 37 ?. Next, 100 ?l of iron probe was added into each well and the solution was mixed well and incubated for 60 min in an oven at 37 °C in the dark. Finally, the OD value was detected with a microplate meter (wavelength?=?593 nm).
Cao et al. used Iron Assay kit ab83366, Lipid Peroxidation (MDA) Assay Kit Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970 and GSH Assay kit GSH Assay Kit (Colorimetric) ab239727 to investigate the effect and mechanism of exosomal miR-26a derived from bone marrow MSCs (BMSCs) on liver fibrosis.
Overexpression of BMSC-derived exosomal miR-26a accelerated ferroptosis in HSCs. Measurement of Fe2+, MDA, and GSH of LX2 cells treated with miR-26a mimic-Exo or NC mimic-Exo.
The level of Fe2+, malonaldehyde (MDA), and glutathione (GSH) was analyzed by using Iron Assay kit (Cat#: ab83366, Abcam, Cambridge, UK), Lipid Peroxidation (MDA) Assay kit (Cat#: Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970, Abcam), and GSH Assay kit (Cat#: GSH Assay Kit (Colorimetric) ab239727, Abcam) based on the operating manual.
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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