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In this web site, technical information about trouble shootings, application examples and
list of reference papers are presented. If you have any questions or any comments, please submit using
QUESTION FORM at the bottom of this page.
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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| Anti 4-HNE monoclonal antibody |
| No. |
Title |
Content |
| 1 |
Applicable species: |
This antibody can be applied to tissue samples from human, rabbit, rat and other animals,
because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue,
blocking of internal mouse IgG will be needed before staining. For example,
Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG. |
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To submit your questions, please click here. |
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| List of references: |
| 1) |
S.Toyokuni, N.Miyake, H.Hiai, M.Hagiwara, S.Kawakishi, T.Osawa and K.Uchida:
The monclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct.
FEBS Lett. 359, p189-191 (1995) |
| 2) |
T.Tanaka, Y.Nishiyama, K.Okada, K.Hirota, M.Matsui, J.Yodoi, H.Hiai, and S.Toyokuni:
Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: independence of tubular necrosis and
sulfhydryl depletion.
Lab. Invest. 77(2), p145-155 (1997) |
| 3) |
Y.Hattori, C.Nishigori, T.Tanaka, K.Uchida, O.Nikaido, T.Osawa, H.Hiai, S.Imamura, and S.Toyokuni:
8-Hydroxy-2'deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure.
J. Invest. Dermatol. 107, p733-737 (1997) |
| 4) |
S.Toyokuni:
Reactive oxygen species-induced molecular damage and its application in pathology.
Pathol. Internat. 49, p91-102 (1999) |
| 5) |
S.Kondo, S.Toyokuni, Y.Iwasa, T.Tanaka, H.Onodera, H.Hiai and M.Imamura:
Persistent oxidative stress in human colorectal carcinoma, but not in adenoma.
Free Rad. Biol. Med. 27, p401-410 (1999) |
| 6) |
T.D. Oberley, S.Toyokuni, and L.I.Szweda:
Localization of hydroxynonenal protein adducts in normal human kidney and selected human kidney cancers.
Free Rad. Biol. Med. 27, p695-703 (1999) |
| 7) |
Hiroko Kimura, Masahiro Mukaida, Kyoko Kuwabara, Teruyo Ito, Kimikazu Hashino, Koji Uchida,
Kazuko Matsumoto and Ken-ichi Yoshida:
4-Hydroxynonenal modifies IgA in rat intestine after lipopolysaccharide injection.
Free Radical Biology and Medicine, 41(6), p973-978 (2006) |
| 8) |
Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T:
Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia
in mice brain.
Stroke. 36(10):p2220-2225(2005) |
| 9) |
Fiorella Biasi, Barbara Vizio, Cinzia Mascia, Ezio Gaia, Neven Zarkovic, Elena Chiarpotto,
Gabriella Leonarduzzi and Giuseppe Poli :
c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction
between transforming growth factor-β1 and 4-hydroxynonenal in colon mucosa
Free Radical Biology and Medicine 41(3), p443-454(2006) |
| 10) |
Isabella Dalle-Donne, Marina Carini, Giulio Vistoli, Luca Gamberoni, Daniela Giustarini,
Roberto Colombo, Roberto Maffei Facino, Ranieri Rossi, Aldo Milzani and Giancarlo Aldini:
Actin Cys374 as a nucleophilic target of α,β-unsaturated aldehydes
Free Radical Biology and Medicine 42(5), p583-598(2007) |
| 11) |
Shintaro Kodai, Shigekazu Takemura, Yukiko Minamiyama, Seikan Hai, Satoshi Yamamoto, Shoji Kubo,
Yasukazu Yoshida, Etsuo Niki, Shigeru Okada, Kazuhiro Hirohashi, Shigefumi Suehiro:
S-allyl cysteine prevents CCl4-induced acute liver injury in rats.
Free Radical Research 41(4), p489-497(2007) |
| 12) |
Tai-Ho Hung, Jeremy N. Skepper, and Graham J. Burton:
In Vitro Ischemia-Reperfusion Injury in Term Human Placenta as a Model for Oxidative Stress in Pathological Pregnancies.
American Journal of Pathology 159(3), p1031-1043(2001)
Anti 4-HNE antibody is applied to immunohistochemistry and western blotting of human placenta. |
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| Hexanoyl-Lys (HEL) ELISA kit |
| No. |
Title |
Contents |
| 1 |
Animals applicable |
HEL ELISA kit is expected to be applied to human, rabbit, rat and other experimental animals,
because lipid oxidation is common to many species. Until today, human urine, dog urine and cat urine are tested.
We would like to share any information about oxidative stress research with researchers, and would like to
contribute to your research. If you have any experiences or any ideas for HEL assay, please don't hesitate to
submit to our web site (biotech@jaica.com).
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| 2 |
Application to human urine: |
Dilution (x 4) with PBS (pH7.4) is recommended.
Concentraion of HEL in normal human urine is expected to be:
Mean 105.4 +/- 67.0 nmol/L.
Mean 85.1 +/- 61.8 pmol/mg creatinine.
Mean 77.2 +/- 54.9 pg/hour kg body weight. |
| 3 |
Application to human serum: |
Sample pretreatment by protease is needed to detect HEL in serum samples.
[Sample pretreatment procedure]
1) Mix 1 volume of serum with 1 volume of PBS (pH7.4).
2) Prepare enzyme solution (14 mg/mL of alpha-chymotrypsin in PBS (pH7.4)).
3) Add 60 uL of enzyme solution to 300 uL of diluted serum.
4) Incubate at 37 degree C overnight (10-18 hours).
5) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove
protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
6) Collect the filtrate and apply to HEL ELISA.
[Assay example]
HEL concentration in normal human serum: 7.96 +/- 4.32 nmol/L. |
| 4 |
Application to animal samples: |
1) Dog urine:
x 4 dilution by PBS is recommended. Assay example 14-58 nmol/L.
2) Cat urine:
x 10 dilution by PBS is recommended. Assay example 108-298 nmol/L.
3) Rat serum:
The same protorol as that for human serum. Assay example 25.8 nmol/L. |
| 5 |
Detection of HEL in LDL: |
HEL can be detected in oxidized LDL. Sample pretreatment is needed.
[Sample pretreatment procedure]
1) Dialyze purified LDL or oxidized LDL to PBS(pH7.4).
2) Add 100 uL of Trypsin (40mg/mL in PBS) and 2 uL of CaCl2solution (100mM) to 100 uL of samples.
3) Incubate at 37 degree C overnight (10-18 hours).
4) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove
protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
5) Collect the filtrate and apply to HEL ELISA.
[Assay example]
HEL level in native human LDL=13.6 nmol/L, oxidized human LDL=363.6 nmol/L |
| 6 |
Application to tissue samples: |
HEL is expected to be detected by follwoing procedure.
[Sample pretreatment procedure]
1) Prepare tissue homogenate in PBS containing 0.1% BHT (butylated hydroxytoluene).
2）Centrifuge, and collect the supernatant.
3) Prepare enzyme solution (14 mg/mL of alpha-chymotrypsin in PBS (pH7.4)).
4) Add 60 uL of enzyme solution to 300 uL of the supernatant.
5) Incubate at 37 degree C overnight (10-18 hours).
6) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove
protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
7) Collect the filtrate and apply to HEL ELISA.
NOTE) BHT may be difficult to dissolve in PBS. Please prepare BHT / ethanol solution, and add BHT solution to
PBS just before start preparing homogenate. |
| 7 |
Application to cultured cells: |
HEL can be detected in cultured cells by follwoing procedure.
[Sample pretreatment procedure]
1) Culture the cells to reach confluent in 6 wells plate.
2) Exchange the medium to serum free.
3) Wash the cells by PBS for 3 times.
4) Scrape the cells in 600 uL of PBS (pH7.4).
5) Add 0.1% BHT (butylated hydroxytoluene), and prepare cell lysate by sonication.
6) Centrifuge, and collect the supernatant.
7) Add 60 uL of enzyme solution to 300 uL of the supernatant.
8) Incubate at 37 degree C overnight (10-18 hours).
9) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove
protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
10) Collect the filtrate and apply to HEL ELISA.
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| 8 |
How to prepare standard curve: |
Any algorithm function can be used alternatively to calculate the results if it is an approximate
semi-logarithm curve (Absorbance=Antilogarithm, Concentration=Logarithm).
Especially since recent micro plate readers have useful functions to draw the standard curves, you can use it.
The semi-logarithm line graph is also enough to use as an approximate function. |
| 9 |
Partial use of the ELISA kit: |
The ELISA kit is made up for 8wells * 12 columns, therefore can be divided accordingly to meet
the number of samples being measured. Please be aware that a new calibration curve is necessary for each measurement
of samples. For a calibration curve 6 levels* 3=18 wells are needed. Almost all samples remain stable when stored
at lower than -20C. Therefore more samples can be measured when you test all of them at the same time. |
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To submit your questions, please click here. |
| List of references: |
| 1) |
Y. Kato, Y. Mori, Y. Makino, Y. Morimitsu, S. Hiroi, T. Ishikawa, T. Osawa.:
Formation of N-ipsyron-(hexanonyl) lysine in protein exposed to lipid hydroperoxide.
J. Biol. Chem. 274(29) p20406-20414 (1999) |
| 2) |
Y. Kato, T. Osawa:
Detection of lipid hydroperoxide-derived protein modification with polyclonal antibodies.
Methods in Molecular Biology, vol. 186, Oxidative stress biomarkers and antioxidant protocols, D Armstrong, Ed.,
Human Press Inc., NJ, USA, p37-44 |
| 3) |
Y. Kato, Y. Miyake, K. Yamamoto, Y. Shimomura, H. Ochi, Y. Mori, T. Osawa:
Preparation of a monoclonal antibody to Nε-(hexanonyl) lysine: applycation to the evaluation of protective effects of
flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.
Biochem. Biophys. Res. Commun. 274(2), p389-393 (2000) |
| 4) |
M. Naito, Wu X, H. Nomura, M. Kodama, Y. Kato, Y. Kato, T. Osawa:
The protective effects of tetrahydrocurcumin on oxidative stress in cholesterol-fed rabbits.
J Atheroscler Thromb. 9(5), p243-250 (2002) |
| 5) |
H. Arai, Y. Kato, K. Fukunaga, S. Mohri, and K. Nakamura:
Formation of Nepsilon-(hexanonyl)lysine in oxidized human very-low density lipoprotein.
J. Electrophoresis 48, p37-40 (2004) |
| 6) |
Y. Kato, A. Yoshida, M. Naito, Y. Kawai, K. Tsuji, M. Kitamura, N. Kitamoto, and T. Osawa:
Identification and Quantification of N-epsilon-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry.
Free Radic. Bio.l Med. 37(11), p1864-1874 (2004) |
| 7) |
T. Osawa, Y. Kato:
Protective role of antioxidative food factors in oxidative stress caused by hyperglycemia.
Ann N Y Acad Sci. 1043, p440-451 (2005) |
| 8) |
K. Minato, M. Gono,H. Yamaguchi, Y. Kato, T. Osawa:
Accumulation of Nepsilon-(Hexanoyl)lysine, an oxidative stress biomarker, in rice seeds during storage.
Biosci Biotechnol Biochem. 69(9), p1806-1810 (2005)
Rice is reported to be oxidized during storage, to form HEL. |
| 9) |
Ryo K, Yamada H, Nakagawa Y, Tai Y, Obara K, Inoue H, Mishima K, Saito I:
Possible involvement of oxidative stress in salivary gland of patients with Sjogren's syndrome.
Pathobiology. 73(5):252-60.(2006)
HEL can be detected in saliva samples from patients with Sjogren's syndrome. |
| 10) |
Suzuki T, Kazui T, Yamamoto S, Washiyama N, Ohkura K, Ohishi K, Bashar AH, Yamashita K,
Terada H, Suzuki K, Akuzawa S, Fujie M:
Effect of prophylactically administered edaravone during antegrade cerebral perfusion in a canine model of old cerebral infarction.
J Thorac Cardiovasc Surg.133(3),p710-716(2007)
Application to canine serum samples. |
| 11) |
Shimizu K, Ogawa F, Akiyama Y, Muroi E, Yoshizaki A, Iwata Y, Komura K, Bae S, Sato S:
Increased Serum Levels of N(epsilon)-(hexanoyl)lysine, A New Marker of Oxidative Stress, in Systemic Sclerosis.
J Rheumatol. 2008 Sep 1.
Serum HEL concentration from systemic sclerosis is higher than that from healthy subjects. |
| 12) |
Tamura H, Takasaki A, Miwa I, Taniguchi K, Maekawa R, Asada H, Taketani T, Matsuoka A, Yamagata Y,
Shimamura K, Morioka H, Ishikawa H, Reiter RJ, Sugino N,
Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate.
J Pineal Res 44(3)p280-287(2008)
Intrafollicular concentration of HEL was significantly reduced by these antioxidant treatment. |
| 13) |
Sakamoto Y, Ishikawa T, Kondo Y, Yamaguchi K, Fujisawa M,
The assessment of oxidative stress in infertile patients with varicocele.
BJU Int. 101(12)p1547-1552(2008)
Azoospermic and oligospermic patients had a significantly higher HEL concentration in seminal plasma. |
| 14) |
Kageyama Y, Takahashi M, Nagafusa T, Torikai E, Nagano A,
Etanercept reduces the oxidative stress marker levels in patients with rheumatoid arthritis. Rheumatol Int. 28(3)p245-251(2008)
Urinary HEL level was reduced at 3 and 6 months after the initial treatment with etanercept. |
| 15) |
Rummenie VT, Matsumoto Y, Dogru M, Wang Y, Hu Y, Ward SK, Igarashi A, Wakamatsu T, Ibrahim O, Goto E,
Luyten G, Inoue H, Saito I, Shimazaki J, Tsubota K,
Tear cytokine and ocular surface alterations following brief passive cigarette smoke exposure. Cytokine. 43(2),p200-208(2008)
HEL concentration in tear was increased by brief passive exposure to cigarette smoke. |
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| Anti Hexanoyl-Lys (HEL) antibody |
| No. |
Title |
Content |
| 1 |
Applicable species: |
This antibody can be applied to tissue samples from human, rabbit, rat and other animals,
because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue,
blocking of internal mouse IgG will be needed before staining. For example,
Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG. |
| 2 |
Immunohistochemistry: |
Protocol can be available at JaICA web site.
Application of anti HEL antibody to HUVECs
Maeda R, et.al.:Hypertens Res 31(1)p141-151(2008) |
| 3 |
Western blottings: |
Anti HEL antibody can be applied to western blottings. Multiple bands will be detected
because HEL adducts can be formed in any proteins which have lysine residues.
[Reference]: H. Arai, Y. Kato, K. Fukunaga, S. Mohri, and K. Nakamura:
Formation of Nepsilon-(hexanonyl)lysine in oxidized human very-low density lipoprotein.
J. Electrophoresis 48, p37-40 (2004)
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|
|
To submit your questions, please click here. |
| List of references: |
| 1) |
Y. Kato, Y. Mori, Y. Makino, Y. Morimitsu, S. Hiroi, T. Ishikawa, T. Osawa.:
Formation of N-epsilon-(hexanonyl) lysine in protein exposed to lipid hydroperoxide.
J. Biol. Chem. 274(29) p20406-20414 (1999) |
| 2) |
Y. Kato, Y. Miyake, K. Yamamoto, Y. Shimomura, H. Ochi, Y. Mori, T. Osawa:
Preparation of a monoclonal antibody to N-epsilon-(hexanonyl) lysine: applycation to the evaluation of protective effects of flavonoid
supplementation against exercise-induced oxidative stress in rat skeletal muscle.
Biochem. Biophys. Res. Commun. 274(2), p389-393 (2000) |
| 3) |
H. Arai, Y. Kato, K. Fukunaga, S. Mohri, and K. Nakamura:
Formation of N-epsilon-(hexanonyl)lysine in oxidized human very-low density lipoprotein.
J. Electrophoresis 48, p37-40 (2004) |
| 4) |
Tomaru M, Takano H, Inoue K, Yanagisawa R, Osakabe N, Yasuda A, Shimada A, Kato Y, Uematsu H:
Pulmonary exposure to diesel exhaust particles enhances fatty change of the liver in obese diabetic mice.
Int J Mol Med. 19(1):17-22 (2007) |
| 5) |
Fukuchi Y, Miura Y, Nabeno Y, Kato Y, Osawa T, Naito M:
Immunohistochemical detection of oxidative stress biomarkers, dityrosine and N(epsilon)-(hexanoyl)lysine, and C-reactive protein
in rabbit atherosclerotic lesions. J Atheroscler Thromb 15(4)p185-192(2008)
HEL was detected in rabbit atherosclerotic lesions. |
| 6) |
Maeda R, Noiri E, Isobe H, Homma T, Tanaka T, Negishi K, Doi K, Fujita T, Nakamura E:
A water-soluble fullerene vesicle alleviates angiotensin II-induced oxidative stress in human umbilical venous endothelial cells.
Hypertens Res 31(1)p141-151(2008)
Application to cultured cells (HUVECs). |
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| Anti Dibromotyrosine (DiBrY) antibody |
| No. |
Title |
Content |
| 1 |
Applicable species: |
This antibody can be applied to tissue samples from human, rabbit, rat and other animals,
because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue,
blocking of internal mouse IgG will be needed before staining. For example,
Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG. |
|
|
To submit your questions, please click here. |
| List of references: |
| 1) |
Wu W, Chen Y, d'Avignon A, Hazen SL.
3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for
eosinophil-dependent tissue injury in vivo.
Biochemistry 38(12), p3538-3548(1999) |
| 2) |
Kato Y, Kawai Y, Morinaga H, Kondo H, Dozaki N, Kitamoto N, Osawa T.
Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine.
Free Radic Biol Med. 38(1), p24-31(2005) |
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| Anti Dityrosine (DT) antibody |
| No. |
Title |
Content |
| 1 |
Applicable species: |
This antibody can be applied to tissue samples from human, rabbit, rat and other animals,
because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue,
blocking of internal mouse IgG will be needed before staining. For example,
Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG. |
|
|
To submit your questions, please click here. |
| List of references: |
| 1) |
Kato Y, Wu X, Naito M, Nomura H, Kitamoto N, Osawa T.
Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody.
Biochem Biophys Res Commun. 275(1), p11-15 (2000). |
| 2) |
Kato Y, Kitamoto N, Kawai Y, Osawa T.
The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine.
Free Radic Biol Med. 2001 Sep 1;31(5):624-32. |
| 3) |
Matsuzaki M, Hasegawa T, Takeda A, Kikuchi A, Furukawa K, Kato Y, Itoyama Y.
Histochemical features of stress-induced aggregates in alpha-synuclein overexpressing cells.
Brain Res. 1004(1-2),p83-90(2004) |
| 4) |
Atwood CS, Perry G, Zeng H, Kato Y, Jones WD, Ling KQ, Huang X, Moir RD, Wang D, Sayre LM, Smith MA,
Chen SG, Bush AI.
Copper mediates dityrosine cross-linking of Alzheimer's amyloid-beta.
Biochemistry. 43(2), p560-568 (2004) |
| 5) |
Shamoto-Nagai M, Maruyama W, Kato Y, Isobe K, Tanaka M, Naoi M, Osawa T.
An inhibitor of mitochondrial complex I, rotenone, inactivates proteasome by oxidative modification and induces aggregation of
oxidized proteins in SH-SY5Y cells.
J Neurosci Res. 74(4), p589-597 (2003) |
| 6) |
Son TG, Zou Y, Yu BP, Lee J, Chung HY.
Aging effect on myeloperoxidase in rat kidney and its modulation by calorie restriction.
Free Radic Res. 39(3), p283-289 (2005) |
| 7) |
Son TG, Zou Y, Jung KJ, Yu BP, Ishigami A, Maruyama N, Lee J.
SMP30 deficiency causes increased oxidative stress in brain.
Mech Ageing Dev. 127(5), p451-457 (2006) |
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| Test kit for Potential antioxidant (PAO) |
| No. |
Title |
Content |
| 1 |
Application to human serum. |
Q: How to store serum samples for PAO assay?
A: Please prepare fresh serum or fresh plasma. Please store frozen below -20 degree C,
because some antioxidants such as ascorbic acid, uric acid and coenzyme Q10 is unstable.
Q: Are plasma samples applicable for PAO assay?
A: Plasma samples with heparin can be applied, but EDTA-plasma can't.
According to our reearch, the mean value of antioxidant level measured by PAO assay is 1069 micro mol/L. |
| 2 |
Application to urine samples. |
PAO assay can be applied to urine samples. But please take care that is the concentration of
uric acid is over 2 mmol/L, dilution by distilled water will be needed. The significance of antioxidants in
urine is unknown. |
| 3 |
Application to food samples: |
1) Red wines: dilute x 8 by distilled water before assay (assay example, 45,479 micro mol/L).
2) Japanese sake: dilute x1 to x8 by distilled water before assay (assay example, 18 to 211 micro mol/L).
3) Black tea: dilute x 8 by distilled water before assay.
4) Coffee: dilute x 28 by distilled water before assay.
5) Green tea: dilute x 8 or more by distilled water before assay (assay example, 8,728 to 60,816 micro mol/L)
|
| 4 |
Antioxidants detectable. |
PAO assay can detect at least follwoing substances:
ascorbic acid, trolox, vitamin E, glutathione, bililubin, uric acid, cathecin, beta-carotene and other polyphenols. |
| 5 |
Preparation of standard. |
Q: Can't draw standard curve.
A: Uric acid powder is used as standard. Please take care to dissolve uric acid completely before use.
The volume of distilled water used is different between vials. Please confirm the volume printed on the label of standard.
Q: How to store dissolved standard?
A: Standard solution (2mM uric acid) can be stored frozen below -20°C. Avoid freeze / thaw cycles.
Q: To calculate antioxidant capacity, why multiply 2189 to uric acid equivalent concentration?
A: To convert from uric acid equivalent to reducing power of copper ion. |
| 6 |
Assay example for ovarian endometrioma: |
Antioxidant power (PAO) can be assessed in ovarian endometrioma.
Ovarian endometrioma: 1197 umol/L
Serous Cystadenoma: 672 umol/L
Mucinous cystadenoma: 424 umol/L
Reference: S Fujii, 31th meeting of Japan BioIron Sociery, Abstruct in Japanese p9-10 (2007) |
|
|
To submit your questions, please click here. |
| List of references: |
| 1) |
Oxidative stress and its association with coronary artery disease and different atherogenic
risk factors.
C VASSALLE, L PETROZZI , N BOTTO, MG ANDREASSI and GC ZUCCHELLI.
Journal of Internal Medicine, Vol.256, p308-315 (2004) |
| 2) |
Vitamin E-coated dialyzers reduce oxidative stress related proteins and markers in hemodialysis ?
a molecular biological approach.
LA Calo, A Naso, E Pagnin, PA Davis, M Castoro, R Corradin, P Riegler, C Cascone, W Huber and A Piccoli
Clinical Nephrology, Vol.62(5), p355-361 (2004) |
| 3) |
Oxidative stress-related factors in Bartter's and Gitelman9s syndrome: relevance for
angiotensin IIsignalling.
Calo LA, Pagnin E, Davis PA, Sartori M, Semplicini A.
Nephrol Dial Transplant ,Vol.18(8) p1518-25 (2003) |
| 4) |
Effect of epoetin on HO-1 mRNA level and plasma antioxidants in hemodialysis patients.
Calo LA, Stanic L, Davis PA, Pagnin E, Munaretto G, Fusaro M, Landini S, Semplicini A, Piccoli A.
Int. J Clin. Ther, Vol.41(5), p187-92 (2003) |
| 5) |
Restored Antioxidant Capacity Parallels the Immunologic and Virologic Improvement in
Children with Perinatal Human Immunodeficiency Virus Infection Receiving Highly Active Antiretroviral Therapy.
M Martino, F Chiarelli, M Moriondo, M Torello, C Azzari, and L Galli
Clinical Immunology, Vol.100(1),p82-6 (2001) |
| 6) |
Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease:
A pilot study.
E Strafacea, P Matarresea, L Gambardella, R Vona, A Sgadari,MC Silveri, W Malorni
FEBS Letters 579, p2759-766(2005)
Serum antioxidant power is lower in patient with Alzheimer disease in comparison with that in control subjects. |
| 7) |
Antioxidant capacity as a reliable marker of stress in dairy calves transported by road
P Pregel, E Bollo, FT Cannizzo, B Biolatti, E Contato, and PG Biolatti
Veterinary Record 156, p53-54 (2005) |
| 8) |
Effetcts of the daily administration of a rehydrating supplement to trotter horses.
A Falaschini, G Marangoni, S Rizzi and MF Trombetta
J Equine Sci 16(1),p1-9 (2005)
Application to horse serum samples. |
| 9) |
Effects of kakisu (persimmon vinegar) on plasma antioxidant power and urinary 8-isoprostane level.
[Article in Japanese]
Mure K, Takeshita T, Morioka I, Arita M
Nippon Eiseigaku Zasshi. 62(1),p32-38 (2007)
Randomized cross over study. Kakisu is one of the Japanese traditional vinegar made from persimmon,
and contains various polyphenols and minerals. After drinking vinegar 20mL / day for 56 days, serum antioxidant power was
significantly higher than control (1447 +/- 36.3 uM vs 1315.9 +/- 31.8 uM, p<0.001). |
| 10) |
Antioxidant effects of flavonoids used as food additives (purple corn color, enzymatically modified
isoquercitrin, and isoquercitrin) on liver carcinogenesis in a rat medium-term bioassay.
Yokohira M, Yamakawa K, Saoo K, Matsuda Y, Hosokawa K, Hashimoto N, Kuno T, Imaida K
J Food Sci. 73(7)C561-568(2008)
Application to rat serum. Serum antioxidant power from rats administrated flavonoids was significantly
higher than that from control. |
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| Anti Acrolein (ACR) antibody |
| No. |
Title |
Content |
| 1 |
Application to 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 micro g/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 |
How to obtain purified acrolein? |
Acrolein is available from Sigma-Aldrich.
Aldrich,
code#110221 (Acrolein) |
|
|
To submit your questions, please click here. |
| 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) |
|
|
| Anti Malondialdehyde (MDA) antibody |
| No. |
Title |
Content |
| 1 |
Application to 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 micro g/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.
|
|
|
To submit your questions, please click here. |
| 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) |
|
|
| Anti Methyl glyoxal (MG) antibody |
| No. |
Title |
Content |
| 1 |
Application to 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 micro g/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 |
Application to 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) |
|
|
To submit your questions, please click here. |
| 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)
|
|
|
| Anti Crotonaldehyde (CRA) antibody |
| No. |
Title |
Content |
| 1 |
Application to 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 micro g/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.
|
|
|
To submit your questions, please click here. |
| 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) |
|
|
| Anti 4-hydroxy-2-hexenal (4-HHE) antibody |
| No. |
Title |
Content |
| 1 |
Application to 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 micro g/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.
|
|
|
To submit your questions, please click here. |
| 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) |
|
|
| Anti 7-ketocholesterol (7-KC) antibody |
| No. |
Title |
Content |
| 1 |
Application to 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 micro g/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 |
Requirement for EDTA during hydrogen peroxide 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 |
About dilution of 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 micro ｍeter thick. |
|
|
To submit your questions, please click here. |
| 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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