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Technical information for oxidative stress biomarkers.  
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Type Product name / code Click below
DNA
oxidation
8-OHdG Check ELISA (Code. KOG-200SE / KOG-200TE / KOG-HS10E) Tech Info References
Anti 8-OHdG monoclonal antibody (N45.1) (Code: MOG-020P / MOG-100P) Tech Info References
Anti Thymidine Glycol (TG) monoclonal antibody (Code: MTG-100P) Tech Info References
Lipid
oxidation
Hexanoyl-Lysine (HEL) ELISA kit (Code: KHL-700E) Tech Info References
Anti Hexanoyl-Lysine (HEL) monoclonal antibody (Code: MHL-021P / MHL-020P) Tech Info References
Anti 4-HNE monoclonal antibody (HNEJ2) (Code: MHN-020P / MHN-100P) Tech Info References
Anti Acrolein (ACR) monoclonal antibody (5F6) (Code: MAR-020n / MAR-100n) Tech Info References
Anti MDA monoclonal antibody (1F83) (Code: MMD-030n) Tech Info References
Anti 4-hydroxy-2-hexenal (4-HHE) monoclonal antibody (Code: MHH-030n) Tech Info References
Anti Crotonaldehyde (CRA) monoclonal antibody (Code: MCA-030n) Tech Info References
Anti Methylglyoxal (MG) monoclonal antibody (Code: MMG-030n) Tech Info References
Anti 7-ketocholesterol (7-KC) monoclonal antibody (Code: MKC-020n) Tech Info References
Protein
oxidation
Dityrosine (DT) ELISA (Code: KDT-010E) Tech Info References
Anti Dityrosine (DT) monoclonal antibody (Code: MDT-020P) Tech Info References
Anti Dibromo-Tyrosine (DiBrY) monoclonal antibody (Code: MBY-020P) Tech Info References
Antioxidant
assay
Test kit for Potential Antioxidant(PAO) (Code: KPA-050) Tech Info References
Traceelements Assay Fe / Iron Assay Kit (ferrozine) (Code: CFE-005) Tech Info References
Fe / Iron Assay Kit (Nitroso-PSAP) (Code: CFE-010) Tech Info References
UIBC Assay Kit (Bathophenanthroline) (Code: CBC-800) Tech Info References
Cu / Copper Assay Kit (3,5-DiBr-PAESA) (Code: CCU-400) Tech Info References
Zn / Zinc Assay Kit (5-Br-PAPS) (Code: CZN-001) Tech Info References
Ca / Calsium Assay Kit (Chlorophosphonazo III) (Code: CCA-020) Tech Info References
Ca / Calsium Assay Kit (o-Cresolphtalein-Complexone) (Code: CCA-030) Tech Info References
Mg / Magnesium Assay Kit (Xyridil Blue-I) (Code: CMG-035) Tech Info References
Anti Acrolein (ACR) antibody
No. Title Content
1 Immunohistochemistry: 1) Paraffin sections were deparaffinized and rehydrated.
2) Sections were quenched for several minutes with 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.
3) Sections were incubated overnight at 4°C with a antibody at a dilution of 1 µg/mL.
4) Antibody binding was visualized by the avidin-biotin-immuno peroxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.
5) 3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.
6) Hematoxylin or Eosin was used as the counter stain.

Technical notes:
1) Sections from which the primary antibodies were omitted served as negative reaction controls.
2) For frozen materials, samples were fixed in 10% formalin, immersed in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan), and stored at -80°C.
3) For paraffin embedded materials, samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and stored at room temperature.
4) Microwave treatment improves the target staining, whereas it declines non-specific background.
5) Protease K treatment declines the target staining.

Positive control:
Aortic wall in the renal tissue of diabetic nephropathy or aortic wall of atherosclerosis.
2 Purified acrolein: Acrolein is available from Sigma-Aldrich.
Aldrich, code#110221 (Acrolein)
3 Western blotting: Modification by acrolein, a component of tobacco smoke and age-related oxidative stress, mediates functional impairment of human apolipoprotein E.
Tamamizu-Kato S, et. al. Biochemistry. 46(28), p8392-8400(2007)

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List of references:
1) Protein-bound acrolein: Potential markers for oxidativestress.
K.Uchida, M.Kanematsu, K.Sakai, T.Matsuda, N.Hattori,Y.Mizuno, D.Suzuki,T.Miyata, N.Noguchi, E.Niki,T.Osawa
Proc.Natl.Acad.Sci.USA,95,4882-4887(1998)
2) Protein-bound acrolein: A novel markers of oxidativestress in Alzheimer's Disease.
Noel Y. Calingasan, Koji Uchida, and Gary E.Gibson
Journal of Neurochemistry.72(2),751-756(1999)
3) A 1-Hour Enzyme-Linked Immunosorbent Assay for Quantitation of Acrolein- and Hydroxynonenal- Modified Proteins by Epitope-Bound CaseinMatrix Method.
K.Satoh,S.Yamada,Y.Koike,Y.Igarashi,S.Toyokuni,T.Kumano,T.Takahata,S.Tsuchida and K.Uchida
Analytical Biochemistry.270,323-328(1999)
4) Red blood cells inhibit activation-induced cell death and oxidative stress in human peripheral blood T lymphocytes.
Fonseca AM, Porto G, Uchida K, Arosa FA.
Blood. 97(10), p3152-60 (2001)
5) Thiolation of protein-bound carcinogenic aldehyde. An electrophilic acrolein-lysine adduct that covalently binds to thiols.
Furuhata A, Nakamura M, Osawa T, Uchida K.
J Biol Chem. 277(31), p27919-27926 (2002)
6) Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis.
Kamelija Zarkovic, Koji Uchida, Danijela Kolenc, Ljiljana Hlupic, Neven Zarkovic
Free Radical Research, 40(6), p543 - 552 (2006)
7) Modification by acrolein, a component of tobacco smoke and age-related oxidative stress, mediates functional impairment of human apolipoprotein E.
Tamamizu-Kato S, Wong JY, Jairam V, Uchida K, Raussens V, Kato H, Ruysschaert JM, Narayanaswami V:
Biochemistry. 46(28), p8392-8400(2007)
Western blotting of ACR-treated ApoE protein.

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Anti Malondialdehyde (MDA) antibody
No. Title Content
1 Immunohistochemistry: 1) Paraffin sections were deparaffinized and rehydrated.
2) Sections were quenched for several minutes with 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.
3) Sections were incubated overnight at 4°C with a antibody at a dilution of 1 µg/mL.
4) Antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.
5) 3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.
6) Hematoxylin or Eosin was used as the counterstain.

Technical Note
1) Sections from which the primary antibodies were omitted served as negative reaction controls.
2) For frozen materials, samples were fixed in 10% formalin, immersed in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan), and stored at -80°C.
3) For paraffinembedded materials, samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and stored at room temperature.
4) Microwave treatment improves the target staining, whereas it declines non-specific background.
5) Protease K treatment declines the target staining.

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List of references:
1) Immunochemical detection of a lipofuscin-like fluorophore derivered from malondialdehyde and lysine.
S Yamada, S Kumazawa, T Ishii, T Nakayama, K Itakura, N Shibata, M Kobayashi, K Sakai, T Osawa and K Uchida.
J.Lipid Res. 42, p1187-1196 (2001)
2) Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress.
Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, Forti G, Baldi E
Mol Med. 21,p109-122(2015). doi: 10.2119/molmed.2014.00158.

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Anti Methyl glyoxal (MG) antibody
No. Title Content
1 Immunohistochemistry: 1) Paraffin sections were deparaffinized and rehydrated.
2) Sections were quenched for several minutes with 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.
3) Sections were incubated overnight at 4°C with a antibody at a dilution of 1 µg/mL.
4) Antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.
5) 3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.
6) Hematoxylin or Eosin was used as the counterstain.

Technical Note
1) Sections from which the primary antibodies were omitted served as negative reaction controls.
2) For frozen materials, samples were fixed in 10% formalin, immersed in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan), and stored at -80°C.
3) For paraffinembedded materials, samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and stored at room temperature.
4) Microwave treatment improves the target staining, whereas it declines non-specific background.
5) Protease K treatment declines the target staining.
2 Western blotting: This antibody can be applied to western blottings.
Reference: Argpyrimidine, a blue fluorophore in human lens proteins: high levels in brunescent cataractous lenses.
Padayatti PS, Ng AS, Uchida K, Glomb MA, Nagaraj RH
Invest Ophthalmol Vis Sci. 42(6), p1299-1304. (2001)

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List of references:
1) Protein modification by a Maillard reaction intermediate methylglyoxal. Immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo.
Uchida K, Khor OT, Oya T, Osawa T, Yasuda Y, Miyata T
FEBS Lett. 410(2-3), p313-318. (1997)
2) Immunological evidence for methylglyoxal-derived modifications in vivo.
Farrukh A Shamsi, Andreea Partal, Candase Sady, Marcus A Glomb and Ramanakoppa H Nagaraj
J. Biol. Chem., 273(12), p6928-6936 (1998)
3) Methylglyoxal Modification of Protein -Chemical and Immunochemical Characterization of Methylglyoxal-Arginine Adduct.
T.Oya, N.Hattori, Y.Mizuno, S.Miyata, S.Maeda, T.Osawa and K.Uchida
J. Biol. Chem., 274, 18492-18502 (1999)
4) Advanced glycation and lipidoxidation of the peritoneal membrane: respective roles of serum and peritoneal fluid reactive carbonyl compounds.
Miyata T, Horie K, Ueda Y, Fujita Y, Izuhara Y, Hirano H, Uchida K, Saito A, van Ypersele de Strihou C, Kurokawa K
Kidney Int. 58(1), p425-435.(2000)
5) Argpyrimidine, a blue fluorophore in human lens proteins: high levels in brunescent cataractous lenses.
Padayatti PS, Ng AS, Uchida K, Glomb MA, Nagaraj RH
Invest Ophthalmol Vis Sci. 42(6), p1299-1304. (2001)
6) High concentrations of glucose induce synthesis of argpyrimidine in retinal endothelial cells.
Padayatti PS, Jiang C, Glomb MA, Uchida K, Nagaraj RH
Curr Eye Res. 23(2), p106-115. (2001)
7) Curcumin Inhibits ROS Formation and Apoptosis in Methylglyoxal-Treated Human Hepatoma G2 Cells.
WEN-HSIUNG CHAN, HSIN-JUNG WU and YAN-DER HSUUW
Ann. N.Y. Acad. Sci. 1042: 372-378 (2005)
8) Heat-shock protein 27 is a major methylglyoxal-modified protein in endotherial cells.
Casper G Schalkwijk, Jan van Bezu, Roel C van der Schors, Koji Uchida, Coen DA Stehouwer, Victor WM van Hinsbergh.
FEBS Lett. 580, p1565-1570 (2006)
Erratum to: Heat-shock protein 27 is a major methylglyoxal-modified protein in endotherial cells.
FEBS Lett. 580, p2400 (2006)
9) Methyl glyoxal elevation is associated with oxidative stress in rheumatoid arthritis.
S. Mukhopadhyay, S. Sen, B. Majhi, K. P. Das, M. Kar
Free Radical Research 41(5) p507-514(2007)
10) Oxidative stresses induced by glycoxidized human or bovine serum albumin on human monocytes.
Philippe Rondeau, Nihar Ranjan Singh, Henri Caillens, Frank Tallet and Emmanuel Bourdon
Free Radical Biology and Medicine 45(6), p799-812 (2008)

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Anti Crotonaldehyde (CRA) antibody
No. Title Content
1 Immunohistochemistry: 1) Paraffin sections were deparaffinized and rehydrated.
2) Sections were quenched for several minutes with 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.
3) Sections were incubated overnight at 4°C with a antibody at a dilution of 1 µg/mL.
4) Antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.
5) 3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.
6) Hematoxylin or Eosin was used as the counterstain.

Technical Note
1) Sections from which the primary antibodies were omitted served as negative reaction controls.
2) For frozen materials, samples were fixed in 10% formalin, immersed in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan), and stored at -80°C.
3) For paraffinembedded materials, samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and stored at room temperature.
4) Microwave treatment improves the target staining, whereas it declines non-specific background.
5) Protease K treatment declines the target staining.

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List of references:
1) Endogenous formation of protein adducts with carcinogenic aldehydes.
K Ichihashi, T Osawa, S Toyokuni, K Uchida J Biol Chem 276(26), p23903-23913 (2001)

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Anti 4-hydroxy-2-hexenal (4-HHE) antibody
No. Title Content
1 Immunohistochemistry. 1) Paraffin sections were deparaffinized and rehydrated.
2) Sections were quenched for several minutes with 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.
3) Sections were incubated overnight at 4°C with a antibody at a dilution of 1 µg/mL.
4) Antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.
5) 3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.
6) Hematoxylin or Eosin was used as the counterstain.

Technical Note
1) Sections from which the primary antibodies were omitted served as negative reaction controls.
2) For frozen materials, samples were fixed in 10% formalin, immersed in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan), and stored at -80°C.
3) For paraffinembedded materials, samples were fixed in 10% formalin, dehydrated, embedded in paraffin, and stored at room temperature.
4) Microwave treatment improves the target staining, whereas it declines non-specific background.
5) Protease K treatment declines the target staining.

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List of references:
1) Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids.
Yamada S, Funada T, Shibata N, Kobayashi M, Kawai Y, Tatsuda E, Furuhata A, Uchida K.
J Lipid Res. 45(4), p626-634 (2004)
2) Accumulation of protein-bound 4-hydroxy-2-hexenal in spinal cords from patients with sporadic amyotrophic lateral sclerosis.
Shibata N, Yamada S, Uchida K, Hirano A, Sakoda S, Fujimura H, Sasaki S, Iwata M, Toi S, Kawaguchi M, Yamamoto T, Kobayashi M
Brain Res. 1019(1-2), p170-177 (2004)

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Anti 7-ketocholesterol (7-KC) antibody
No. Title Content
1 Immunohistochemistry: 1) Wash OCT compound with PBS.
2) Frozen sections were quenched for several minutes with 1% EDTA added 3% hydrogen peroxide, rinsed in PBS, pretreated for 30 min with 3% nonimmune animal serum in PBS.
3) Sections were incubated overnight at 4°C with a antibody at a dilution of 10 µg/mL.
4) Antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using the Vectastain ABC kit (Vector, Burlingame, CA) according to the manufacturer's instructions.
5) 3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen.
6) Hematoxylin or Eosin was used as the counterstain.

Technical Note:
1) Frozen materials only.
2) Sections from which the primary antibodies were omitted served as negative reaction controls.
3) For frozen materials, samples were fixed in 10% formalin, immersed in 30% sucrose in PBS, embedded at OCT (Sakura, Tokyo, Japan), and stored at -80°C.
2 Fix conditions: To fix fresh tissue within 5mm thick, 20% formalin for 24 to 48 hours is recommended.
3 EDTA for H2O2 treatment. If Milli-Q water is used, EDTA is not needed. But if EDTA affect your staining results, add 1% EDTA for hydrogen peroxide solution.
4 Blocking of internal avidin. Avidin blocking reagent is available from Vector Laboratories.
5 Secondary antibody: The dilution factor may be different depending on the kit used in your laboratory. For example, dilute x5000 to x10000 rabbit anti-mouse IgG or goat anti-mouse IgG, and incuvate for 2hours at room temperature. Please refer to manufacturer's instructions.
6 The thickness of samples. We recoomend to prepare at 3 to 7 µm thick.
7 Esterified 7-ketocholesteol: Rectivity to esterified 7-KC has not been tested, because esterified 7-KC is highly hydrophobic.

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List of references:
1) Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects.
Free Radical Biology and Medicine, 41(6), p902-910 (2006)
David A. Larsson, Sarah Baird, Jerome Diinga Nyhalah, Xi-Ming Yuan and Wei Li
2) In vivo interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol, potential surrogate markers for oxidative stress.
Free Radical Biology and Medicine 43(5), p695-701(2007)
Hanna Larsson, Ylva Bottiger, Luigi Iuliano and Ulf Diczfalusy

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What is oxidative stress: FAQ
[NOTICE]: Our products are for RESEARCH USE ONLY. Not for diagnostic, medical or other use.

We are making efforts to prevent errors or mistakes on preparing web site documents, instruction manuals and products.
But even if some damage would causedby such faults, we will be exempt from responsibility.
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