Iron Assay kit LS – Ferrozine method | Metallo Assay LS

• Features
• Assay Principle
• Sample Preparation
• Handling Procedure
• Citations
• Product Information

Features

• Using microplate reader for assay, no need to use Atomic Absorption Spectrometry or ICP-OES Optical Emission Spectrometry, which have been mainly used so far.
• Good correlation with the result of Atomic Absorption Spectrometry and ICP-OES.
• High throughput measurement by using microplate reader.
• All animal species sample can be measured.
• Assay time : just 10 minutes!
• 1 point calibration – do not need multiple point measurement to make standard curve (r²=0.9999).
• Kit does not contain any hazardous components like cyanide or azide as preservative.
• Samples : Serum, Plasma, Urine, Saliva, Cell lysate, Tissue extract, Hair extract, Water, etc.
• In combination with UIBC Assay kit LS Bathophenanthroline method (#UIB02AE), TIBC (Total Iron Binding Capacity) can be measured.

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Assay Principle

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Sample Preparation

• Samples which do not require preparation : e.g. Serum, Plasma and Urine
  * EDTA treated sample is not suitable for this assay.
  * Pooled urine should add HCl for storing. When assay, adujust sample pH 2 to 3.


• Samples which require acid extraction : e.g. Cell Lysate, Tissue Homogenate and Other liquid samples
  * Measurable chemical species is limited as follows:
  　　・Free iron
  　　・Iron binds to protein (Protein-Fe2+, e.g. albumin, metallothionein)
  * Lysis buffer contains EDTA is not suitable for this assay.
  * Organoiron and Heme iron requires acidolysis.


• Microwave method and samples require acidolysis : e.g. Samples contain strong chelate agents such as EDTA, Organoiron (in the case of single bond -C-Fe-C-), Iron in cyclic ligand (Fe-Porphyrin)
  * After acidolysis, adjust sample pH in 2 to 3.

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Handling Procedure

(1)Apply DW, Standard and Sample (40 uL) to 96 well plate.
(2)Add R-A buffer (200 uL).
(3)Incubate at room temperature for 5 minutes.
(4)Read the absorbance at 560 nm (main) as OD1.
(5)Add R-R Chelate Color (8 uL).
(6)Incubate at room temperature for 5 minutes.
(7)Read the absorbance at 560 nm (main) as OD2.
(8)Calculate concentration.     ΔODStd = (OD2Std - OD1Std) - (OD2Bl1 - ODBl)     ΔODS = (OD2S - OD1S) - (OD2Bl - OD1Bl)     Iron (ug/dL) = ΔODS/ΔODStd x 200     Iron (uM) = ΔODS/ΔODStd x 35.8	*ODS : OD sample, ODBl : OD blank,    ODStd : OD Standard

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Citations

No.	Literature information	Remarks	URL
1	Kagoya Y et al. JAK2V617F+ myeloproliferative neoplasm clones evoke paracrine DNA damage to adjacent normal cells through secretion of lipocalin-2. Blood 2014 Nov;124(19):2996-3006 Kagoya Y et al 2014/01/01
2	Tajima S et al. Angiotensin II alters the expression of duodenal iron transporters, hepatic hepcidin, and body iron distribution in mice. Eur J Nutr 2014 Aug; Tajima S et al 2014/01/01
3	Tsugawa H et al. FecA1, a bacterial iron transporter, determines the survival of Helicobacter pylori in the stomach. Free Radic. Biol. Med. 2012 Mar;52(6):1003-10 Tsugawa H et al 2012/01/01
4	Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice. Scientific Reports 6; Article number: 22476, 02 March 2016
5	Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3–E3 ubiquitin ligase-dependent pathway Biology; Volume 35, May 2016, Pages 66–76
6	Regulation of iron homeostasis by the p53-ISCU pathway; Sci Rep. 2015; 5: 16497.
7	Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria　Plos One; September 18, 2015
8	Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease　Hypertension Research;38, 463-470 (July 2015)
9	Temporary Dietary Iron Restriction Affects the Process of Thrombus Resolution in a Rat Model of Deep Vein Thrombosis　Plos one; May 11, 2015
10	Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging and hyperthermia treatment 　Theranostics ; April 23, 2013 Hayashi K et al 2013/01/01
11	Takenaka M et al. Iron regulatory protein 2 in ovarian endometrial cysts. Biochem. Biophys. Res. Commun. 2017 Jun;487(4):789-794 Takenaka M et al 2017/01/01
12	Oshima K et al. Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation. Lab. Invest. 2017 May;97(5):555-566 Oshima K et al 2017/01/01
13	Yoshida K et al. Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts. Biochem. J. 2017 Apr;474(8):1347-1360 Yoshida K et al 2017/01/01
14	Watanabe S et al. Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase. Sci Rep 2016 Dec;6:38720 Watanabe S et al 2016/01/01
15	Murotomi K et al. Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice. Sci Rep 2016 Mar;6:22476 Murotomi K et al 2016/01/01
16	Ikeda Y et al. Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway. J Trace Elem Med Biol 2016 May;35:66-76 Ikeda Y et al 2016/01/01
17	Watanabe S et al. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria. PLoS ONE 2015;10(9):e0138434 Watanabe S et al 2015/01/01
18	Naito Y et al. Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease. Hypertens. Res. 2015 Jul;38(7):463-70 Naito Y et al 2015/01/01
19	Oboshi M et al. Temporary dietary iron restriction affects the process of thrombus resolution in a rat model of deep vein thrombosis. PLoS ONE 2015;10(5):e0126611 Oboshi M et al 2015/01/01
20	Ikeda Y et al. Iron chelation by deferoxamine prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction. PLoS ONE 2014;9(2):e89355 Ikeda Y et al 2014/01/01
21	Isobe N et al. TEMPO-oxidized cellulose hydrogel as a high-capacity and reusable heavy metal ion adsorbent. J. Hazard. Mater. 2013 Sep;260:195-201 Isobe N et al 2013/01/01
22	Ikeda Y et al. Dietary iron restriction inhibits progression of diabetic nephropathy in db/db mice. Am. J. Physiol. Renal Physiol. 2013 Apr;304(7):F1028-36 Ikeda Y et al 2013/01/01
23	Nakaya M et al. GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance. Nat Commun 2013;4:1532 Nakaya M et al 2013/01/01

• No.： 1
• Literature information：
  Kagoya Y et al. JAK2V617F+ myeloproliferative neoplasm clones evoke paracrine DNA damage to adjacent normal cells through secretion of lipocalin-2. Blood 2014 Nov;124(19):2996-3006
  Kagoya Y et al
  2014/01/01
  
• Remarks：
  
• URL：
  

• No.： 2
• Literature information：
  Tajima S et al. Angiotensin II alters the expression of duodenal iron transporters, hepatic hepcidin, and body iron distribution in mice. Eur J Nutr 2014 Aug;
  Tajima S et al
  2014/01/01
  
• Remarks：
  
• URL：
  

• No.： 3
• Literature information：
  Tsugawa H et al. FecA1, a bacterial iron transporter, determines the survival of Helicobacter pylori in the stomach. Free Radic. Biol. Med. 2012 Mar;52(6):1003-10
  Tsugawa H et al
  2012/01/01
  
• Remarks：
  
• URL：
  

• No.： 4
• Literature information：
  Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice. Scientific Reports 6; Article number: 22476, 02 March 2016
  
  
• Remarks：
  
• URL：
  

• No.： 5
• Literature information：
  Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3–E3 ubiquitin ligase-dependent pathway Biology; Volume 35, May 2016, Pages 66–76
  
  
• Remarks：
  
• URL：
  

• No.： 6
• Literature information：
  Regulation of iron homeostasis by the p53-ISCU pathway; Sci Rep. 2015; 5: 16497.
  
  
• Remarks：
  
• URL：
  

• No.： 7
• Literature information：
  Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria　Plos One; September 18, 2015
  
  
• Remarks：
  
• URL：
  

• No.： 8
• Literature information：
  Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease　Hypertension Research;38, 463-470 (July 2015)
  
  
• Remarks：
  
• URL：
  

• No.： 9
• Literature information：
  Temporary Dietary Iron Restriction Affects the Process of Thrombus Resolution in a Rat Model of Deep Vein Thrombosis　Plos one; May 11, 2015
  
  
• Remarks：
  
• URL：
  

• No.： 10
• Literature information：
  Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging and hyperthermia treatment 　Theranostics ; April 23, 2013
  Hayashi K et al
  2013/01/01
  
• Remarks：
  
• URL：
  

• No.： 11
• Literature information：
  Takenaka M et al. Iron regulatory protein 2 in ovarian endometrial cysts. Biochem. Biophys. Res. Commun. 2017 Jun;487(4):789-794
  Takenaka M et al
  2017/01/01
  
• Remarks：
  
• URL：
  

• No.： 12
• Literature information：
  Oshima K et al. Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation. Lab. Invest. 2017 May;97(5):555-566
  Oshima K et al
  2017/01/01
  
• Remarks：
  
• URL：
  

• No.： 13
• Literature information：
  Yoshida K et al. Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts. Biochem. J. 2017 Apr;474(8):1347-1360
  Yoshida K et al
  2017/01/01
  
• Remarks：
  
• URL：
  

• No.： 14
• Literature information：
  Watanabe S et al. Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase. Sci Rep 2016 Dec;6:38720
  Watanabe S et al
  2016/01/01
  
• Remarks：
  
• URL：
  

• No.： 15
• Literature information：
  Murotomi K et al. Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice. Sci Rep 2016 Mar;6:22476
  Murotomi K et al
  2016/01/01
  
• Remarks：
  
• URL：
  

• No.： 16
• Literature information：
  Ikeda Y et al. Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway. J Trace Elem Med Biol 2016 May;35:66-76
  Ikeda Y et al
  2016/01/01
  
• Remarks：
  
• URL：
  

• No.： 17
• Literature information：
  Watanabe S et al. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria. PLoS ONE 2015;10(9):e0138434
  Watanabe S et al
  2015/01/01
  
• Remarks：
  
• URL：
  

• No.： 18
• Literature information：
  Naito Y et al. Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease. Hypertens. Res. 2015 Jul;38(7):463-70
  Naito Y et al
  2015/01/01
  
• Remarks：
  
• URL：
  

• No.： 19
• Literature information：
  Oboshi M et al. Temporary dietary iron restriction affects the process of thrombus resolution in a rat model of deep vein thrombosis. PLoS ONE 2015;10(5):e0126611
  Oboshi M et al
  2015/01/01
  
• Remarks：
  
• URL：
  

• No.： 20
• Literature information：
  Ikeda Y et al. Iron chelation by deferoxamine prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction. PLoS ONE 2014;9(2):e89355
  Ikeda Y et al
  2014/01/01
  
• Remarks：
  
• URL：
  

• No.： 21
• Literature information：
  Isobe N et al. TEMPO-oxidized cellulose hydrogel as a high-capacity and reusable heavy metal ion adsorbent. J. Hazard. Mater. 2013 Sep;260:195-201
  Isobe N et al
  2013/01/01
  
• Remarks：
  
• URL：
  

• No.： 22
• Literature information：
  Ikeda Y et al. Dietary iron restriction inhibits progression of diabetic nephropathy in db/db mice. Am. J. Physiol. Renal Physiol. 2013 Apr;304(7):F1028-36
  Ikeda Y et al
  2013/01/01
  
• Remarks：
  
• URL：
  

• No.： 23
• Literature information：
  Nakaya M et al. GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance. Nat Commun 2013;4:1532
  Nakaya M et al
  2013/01/01
  
• Remarks：
  
• URL：
  

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Product Information

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