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Iron Colorimetric Assay Kit

based on 5 citations in multiple journalsIron Colorimetric Assay Kit54.1 4
WARNING: This product can expose you to chemicals including Thiourea, which is [are] known to the State of California to cause cancer.  For more information go to
Catalog #: K390

Product Details

Cat # +Size K390-100
Size 100 assays
Detection Method Absorbance (593 nm)
Species Reactivity Mammalian
Applications To measure ferrous and/or ferric ion in samples
Features & Benefits • Simple, Fast and convenient
• The assay is sensitive and stable
• The kit measures iron in the linear range of 0.4 to 10 nmol or 8 µM to 200 µM iron concentration in various samples.
Kit Components • Iron Assay Buffer
• Iron Probe
• Iron Reducer
• Iron Standard (100 mM)
Storage Conditions -20ºC
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Iron is essential to nearly all known organisms. It is generally stored in the centre of metalloproteins, in the heme complex, and in oxygen carrier proteins. Inorganic iron also contributes to redox reactions in the iron-sulfur clusters of many enzymes, such as nitrogenase and hydrogenase. BioVision's Iron Assay Kit provides a simple convenient means of measuring Ferrous and/or Ferric ion in sample. In the assay, ferric carrier protein will dissociate ferric into solution in the presence of acid buffer. After reduction to the ferrous form (Fe²ᶧ), iron reacts with Ferene S to produce a stable colored complex and give absorbance at 593 nm. A specific chelate chemical is included in the buffer to block copper ion (Cu²ᶧ) interference. The kit measures iron in the linear range of 0.4 to 20 nmol in 50 µl sample, or 8 µM to 400 µM iron concentration in various samples.

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What are normal iron levels?
1) Normal serum Iron ~10-40 µM. 2) 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)
On addition of Iron Probe (provided in kit) we could see an immediate development of color in wells containing the standards; However, even after incubation for longer period (over 1 hr and more) the wells containing our samples did not show any change in color at all.
I suspect that the client is using too dilute a sample to get readings within in the linear range of the standard curve. They most likely will do better if they use higher volumes or more concentrated of the samples.
Could you please help me answer our customer’s inquiry? They are seeing the samples turn cloudy but not blue with addition of the probe. It appears they are doingf everything accordign to protocol (details below). Is it common for the sample/probe to do this?
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 the client add 5 µl/well of 1 M SDS (28.8% or 288 mg/ml of SDS) to all their sample wells after step IV.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).
Customer is looking for any recommended preperation steps for using this kit with serum. For example, leaving the serum at room temp for 2 hours. What about centrifuging? Any tips would be great.
For serum prep: Collect whole blood in a covered test tube on ice. After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. This usually takes 15-30 minutes. Remove the clot by centrifuging at 1,000-2,000 x g for 10 minutes in a refrigerated centrifuge. Following centrifugation, it is important to immediately transfer the serum into a clean polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the serum is not analyzed immediately, the serum should be apportioned into 0.5 ml aliquots, stored, and transported at –80°C. It is important to avoid freeze-thaw cycles because this is detrimental to many serum components. Samples which are hemolyzed, icteric or lipemic can invalidate certain tests. Do not leave it for length of time at RT after preparation.
When I added Iron Probe (NM), samples turned cloudy. To figure it out, I tried several ways. I found if I added acid solution to my samples (ex: HCL), it turned clear. On the contrary, when I added NaOH, it turned more cloudy. Please let me know if you can provide any insight for our customer.
This assay works in an acidic environment. That is why when the client adds the HCl, the samples are turning clear. One of the reasons that the samples are not changing color is because they are very dilute. Please use more of the samples. Ideally the Ferene S to iron ratio should be large than 5. On the other hand, I suspect that there is some interference being generated by some components in the samples leading to the cloudiness. Copper (II) and Cr(III) will interfere with the result, Copper (II) can be block by the thiourea.
I have used this kit with serum samples successfully, but have issues using it with plasma. Why?
This kit is not compatible with plasma samples collected using metal chelators such as EDTA and citrate.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
I like to test cell lysate and would like to know how to collect the cells. Can I digest cells by trypsin or scrape cells off? After cell collection, should I wash the cells with PBS? Could you provide your cell preparation protocol if possible?
We have not tested cells/tissues with this kit. However, the kit should work with a variety of sample types. You can scrape off the cells with a cell scraper. Before you scrape off, please gently wash the cells with PBS. For sample preparation, please use 1x10^6 cells in 100 µl of Iron assay buffer, homogenize on ice using a dounce homogenizer or gentle sonication on ice with a few short pulses. Centrifuge at 10,000g and use the supernatant for the assay.
Bai, T. et al., (2017) Haloperidol, a sigma receptor 1 antagonist, promotes ferroptosis in hepatocellular carcinoma cells, Biochemical and Biophysical Research Communications, Sep.2017, 491(4):919-925
Brown et al., Increased Ferroportin-1 Expression and Rapid Splenic Iron Loss Occur with Anemia Caused by Salmonella enterica Serovar Typhimurium Infection in Mice. Infect. Immun., Jun 2015; 83: 2290 - 2299.
Aijun Qiao et al., MicroRNA-210 Decreases heme Levels by Targeting Ferrochelatase in Cardiomyocytes. JAHA, Apr 2013; 2: e000121.
D. E. Brown et al., Salmonella enterica Causes More Severe Inflammatory Disease in C57/BL6 Nramp1G169 Mice Than Sv129S6 Mice. Veterinary Pathology, Feb 2013; 0.1177/0300985813478213.
Gurugirijha Rathnasamy et al., Iron and Iron Regulatory Proteins in Amoeboid Microglial Cells Are Linked to Oligodendrocyte Death in Hypoxic Neonatal Rat Periventricular White Matter through Production of Proinflammatory Cytokines and Reactive Oxygen/Nitrogen Species, J. Neurosci., Dec 2011; 31: 17982 - 17995.
For more citations of this product click here