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Ferrozine Iron Assay
Last updated date: Mar 12, 2024 Views: 895 Forks: 0
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- Take 50 μL lysate (Take double volume for lysis buffer as control)
- Add 11 μL concentrated HCl (11.6 M)
- Place all tubes on the 95℃ heating block for 20 minutes.
- Centrifuge at the highest speed for 10 min. Remove very gently from centrifuge.
- Remove 45 μL of supernatant very carefully. It is essential not to include any of the pellet in what you remove. These pellets are very delicate.
- Re-centrifuge if needed, especially for the control.
- Add 18 μL ascorbate (75 mM) to each tube. Ascorbate acts as a reductant, moving Fe from the 3+ state to the 2+ state. Aliquots are in Iron Assay box in -20℃. Vortex-quick spin. Incubate for 2 min
- Add 18 μL ferrozine (10 mM) to each tube. Ferrozine acts as an oxidant, taking Fe from the 2+ state to the 3+ state. Aliquots are in Iron Assay box in -20℃. Both ferrozine and ascorbate are light sensitive and can be kept in the fridge for about a week without loss of activity. Incubate for 2 min
- Add 36 μL saturated ammonium acetate (NH4Ac) to each tube. Vortex each tube to mix. Incubate for 2 min
Record spectra (500-700 nm) and read samples at 562 nm
Formula for calculating [Fe] (usually ends up pmoles per µL sample)
[Fe] (M) = (ΔOD562 * 94/77 * 130 / 50) / 27900
[Fe] (pmol/ µL) = (ΔOD562 * 61 / 50 * 117 / 45) / 27900] * 106
Related files
Iron Ferrozine Assay-Manik.doc 
Copyright: © 2024 The Author(s); This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Li, K(2024). Ferrozine Iron Assay. Bio-protocol Preprint. bio-protocol.org/prep2600.
- Xu, T., Cai, J., Wang, L., Xu, L., Zhao, H., Wang, F., Meyron-Holtz, E. G., Missirlis, F., Qiao, T. and Li, K.(2023). Hormone replacement therapy for postmenopausal atherosclerosis is offset by late age iron deposition. eLife. DOI: 10.7554/eLife.80494
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