Overview

  • Product name
    Iron Assay Kit
    See all Iron kits
  • Detection method
    Colorimetric
  • Sample type
    Urine, Serum, Other biological fluids, Tissue Extracts, Cell culture media
  • Assay type
    Quantitative
  • Sensitivity
    > 8 µM
  • Range
    8 µM - 400 µM
  • Assay time
    1h 00m
  • Product overview

    Iron Assay Kit ab83366 provides a simple convenient means of measuring ferrous (Fe2+) and/or ferric (Fe3+) iron in biological samples.


    In the iron assay protocol, ferrous iron (Fe2+) reacts with Ferene S to produce a stable colored complex with absorbance at 593 nm. Ferric iron (Fe3+) can be reduced to form Fe2+ enabling the measurement of total iron (Fe2+ and Fe3+). 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 8 µM to 400 µM.


    Iron assay protocol summary:
    - add samples and standards to wells
    - for Fe2+ assay add assay buffer only, for total iron (Fe2+ and Fe3+) add iron reducer
    - incubate for 30 min at 25ºC
    - add iron probe and incubate for 60 min at 25ºC
    - analyze with microplate reader

  • Platform
    Microplate reader

Properties

Images

  • 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).

  • Iron measured in mouse muscle lysate showing quantity (micrograms) per microgram total protein

  • Iron measured in mouse liver lysate showing quantity (micrograms) per microgram total protein

  • Iron measured in human urine showing concentration (micromolar)

  • 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.

Protocols

References

This product has been referenced in:
  • Meng Y  et al. Changes of lysosomal membrane permeabilization and lipid metabolism in sidt2 deficient mice. Exp Ther Med 16:246-252 (2018). Read more (PubMed: 29896245) »
  • Sui M  et al. Magnesium isoglycyrrhizinate ameliorates liver fibrosis and hepatic stellate cell activation by regulating ferroptosis signaling pathway. Biomed Pharmacother 106:125-133 (2018). Read more (PubMed: 29957462) »
See all 17 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Question
Answer

Unfortunately, this kit is not suitable for use with plasma samples due to the presence of iron binding transferrin in plasma leading to inaccurate measurements of free iron.

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Question
Answer

Yes, you can definitely measure ferrous and ferric acid separately with this kit. Please follow the protocol for measuring the ferrous and the total iron in the samples. When you subtract the ferrous levels from the total level, you get the ferric levels in the samples.

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Answer

This is a common problem seen in liver and serum samples. Lipoproteins in the sample are the main culprits behind this turbidity.
For this purpose, we would recommend that to add 5 µl/well of 1 M SDS (28.8% or 288 mg/ml of SDS) to all their sample wells after step 4.2. Incubate for 30 min at 25 °C as mentioned in the datasheet and then follow the protocol. This SDS will clear up the turbidity (by dissolving any lipoproteins in the samples).

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Question
Answer

We do not typically recommend using RIPA buffer with our kits since it contains SDS which can denature proteins but for this assay it could work. We have not tested with RIPA buffer. We recommend using the assay buffer that comes with the kit.

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Question
Answer

I can confrim that the ab83366 Iron Assay Kit will measure the free ferrous and ferric forms of iron and not the iron complexed in Heme.

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Answer

1. All components should be stored in aliquots according to individual use requirements at -20C to prevent freeze-thaw cycles.

2. The plate can be covered by a plate cover and aluminium foil to protect from light during incubation. The incubation is at RT and hence there is not much concern about evaporation at 25C.

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Answer

Since the specific probe is proprietary we do not have a spectrum to share, but in our experience reading at 570 nm should work and may simply reduce the sensitivity slightly.

The emission profile shows a sharp peak at 580-590 nm, can measure signal within 590 +/- 20 nm.

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Detection of iron levels in hair samples

Average Good 4/5 (Ease of Use)
Abreviews
We used the iron assay kit to evaluate whether iron can be detected in hair shaft samples. In this case, we used a hair sample from a Rhesus primate, since we had a sufficient amount to test. The hair sample was milled into a fine powder, homogenized with assay buffer (4x volume) and centrifuged as directed. We initially tested various small amounts of hair (ranging from 1.8 to 12.1mg), given that these samples are often only available in limited quantities. The resulting absorbance was very close to the base nm of the 0 standard, and translated to -0.043 to 0.034nmoles per 50μl sample. We had used two sets of standard concentrations: the set of standards recommended in the protocol (0-10nmol/well) and a 1:10 dilution of those standards (0-1nmol/well). Both standard curves had an R-value better than 0.98, and the diluted standard curve was used since it best encompassed the resulting nm.
We repeated the assay with a larger amount of hair (55.9mg). In the increased sample we were able to detect 0.14-0.23nmole per 50μl sample, which was closer to the stated range of the kit (0.4-20 nmole/50μl). This indicates that it is possible to detect iron in hair shaft sample using this kit. However, given the relatively large amount of sample needed, it is not an ideal method for limited sample sizes. The kit itself was very easy to use.

Dr. Erilynn Heinrichsen

Verified customer

Submitted Nov 05 2013

Answer

The buffer in this kit should be compatible with generic protein assays. We would prefer using the Bradford method. If the protein cannot be detected that way, we would recommend to have duplicates of the same number of cells. One of the samples can then be used for the protein assay and the second one can be lysed with any generic lysis buffer, so the protein quantity can be estimated.

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Question
Answer

Normal serum Iron ~10-40 µM. Cells such as HeLa or cervical cells typically contain 1-0.9 pg iron/cell so 2 x 106 cells lysed in ~ 250 ul of assay buffer (Fe MW 55.85) should have about 4 – 8 nmol Fe per 50 ul test sample-possibly less if iron tightly bound and not released well be acid buffer (assay buffer)

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1-10 of 21 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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