Ferrozine |
| Catalog No.GC47346 |
Ferrozine reacts with divalent iron to form a stable magenta complex species and used for the direct determination of iron in water with maximum absorbance at 562 nm.
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 69898-45-9
Sample solution is provided at 25 µL, 10mM.
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Ferrozine reacts with divalent iron to form a stable magenta complex species and used for the direct determination of iron in water with maximum absorbance at 562 nm [1,2]. The visible absorption spectrum of the ferrous complex of ferrozine exhibits a single sharp peak at 562 nm. At this wavelength, the molar absorptivity is 27,900 and the Beer-ambert law is obeyed to approximately 4 mg/L of Fe [1].
Ferrozine assay of ISE6 cells[3]
The ferrozine assay for measuring non-haem iron was adapted to determine the concentration of iron in ISE6 cells. After knockdown and/or iron exposure, cells were collected, and cell lysates were collected using the method described above. Concentrated HCl (99.5%) was added and then heated to 95 °C. After cooling to room temperature, the mixture was centrifuged, and the supernatant was obtained, to which was added 75 mM ascorbate or water. Afterward, 10 mM ferrozine was added. Saturated ammonium acetate was added to facilitate colour development. Absorbance was measured at 550 nm, and the iron concentration was calculated based on a molar extinction coefficient of the iron-ferrozine complex of 27,900 M-1cm-1 and based on the protein concentration. The protein concentration was measured using a Micro BCA Protein Assay Kit. The total iron concentration is computed from samples with ascorbate. The ferrous iron concentration was computed from samples without ascorbate (reducing agent), while the ferric iron concentration is computed from the difference between the total iron and ferrous iron concentration.
References:
[1]. Stookey L L. Ferrozine---a new spectrophotometric reagent for iron[J]. Analytical chemistry, 1970, 42(7): 779-781.
[2]. Jeitner T M. Optimized ferrozine-based assay for dissolved iron[J]. Analytical biochemistry, 2014, 454: 36-37.
[3]. Hernandez E P, Kusakisako K, Talactac M R, et al. Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6)[J]. Scientific reports, 2018, 8(1): 1-12.
This plan only provides a guide, please modify it to meet your specific needs.
1. Divide the equally divided sample of cell lysate (100μL) Placed in Eppendorf tube and connected to 100μL 10mM HCl (Fe3+ solvent) and 100μL iron release reagent (dissolved in an equal volume of 1.4M HCl and 4.5% (w/v) KMnO4 in ultrapure water, freshly prepared) mixed;
Note: HCl/KMnO4 pretreatment can quantitatively release iron from proteins, including iron storage protein ferritin and heme proteins such as hemoglobin. The digestion of iron containing proteins mediated by HCl/KMnO4 is essential for the iron quantification of heme proteins. For example, if the protein is not pretreated with an acidic permanganate solution, treating hemoglobin with iron zinc will not result in the recovery of any Fe2+- Ferrozine complexes.
2. Incubate the mixture in a fume hood at 60℃ for 2 hours, as chlorine gas will be generated during the reaction process;
3. After cooling the mixture to room temperature, add 30μL of iron detection reagents (6.5mM Ferrozine, 6.5mM neocuproine, 2.5M ammonium acetate, and 1M ascorbic acid dissolved in water) to each tube;
4. After 30 minutes, remove 280μL from each tube the solution into the well of a 96 well plate and measure the absorbance at 562nm on a microplate reader.
5. 100μL FeCl3 standard (0-300μM) in10 mM HCl, 100μL 50 mM NaOH, 100μL release reagent and 30μL mixture in the detection reagent. Calculate the iron content of the sample by comparing its absorbance with that of an equal volume standard concentration range prepared in a similar manner to the sample.
Notes:
1) Fluorescent dyes all have quenching problems, please try to avoid light as much as possible to slow down fluorescence quenching.
2) For your safety and health, please wear laboratory clothes and disposable gloves when operating.
References:
[1].Jan Riemer, et al. Colorimetric ferrozine based assessment for the quantification of iron in cultured cells 2004 Aug 15; 331 (2): 370-5. doi: 10.1016/j.ab. 204.03.049.
| Cas No. | 69898-45-9 | SDF | |
| Canonical SMILES | OS(C1=CC=C(C2=C(C3=CC=C(S([O-])(=O)=O)C=C3)N=C(C4=CC=CC=N4)N=N2)C=C1)(=O)=O.[Na+] | ||
| Formula | C20H13N4O6S2.Na | M.Wt | 492.5 |
| Solubility | PBS (pH 7.2): 1 mg/ml | Storage | Store at 2-8°C |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
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| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
|
1 mg | 5 mg | 10 mg |
| 1 mM | 2.0305 mL | 10.1523 mL | 20.3046 mL |
| 5 mM | 0.4061 mL | 2.0305 mL | 4.0609 mL |
| 10 mM | 0.203 mL | 1.0152 mL | 2.0305 mL |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
g
μL
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )
Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Quality Control & SDS
- View current batch:
- Purity: >97.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 25 reference(s) in Google Scholar.)
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