Dojindo Oxidative Stress Products - Dojindo Molecular … · · 2013-02-21Dojindo Oxidative...
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Dojindo Oxidative Stress Products
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Dojindo Oxidative Stress Products
Oxidative Stress Research Products - Index
Purpose Product Page
1. Anti Oxidant Detection Measurementofsuperoxidedismutaseactivity SODAssayKit-WST 1
Quantificationoftotalglutathione TotalGlutathioneQuantificationKit 4 Separatequantificationofreduced(GSH)and oxidized(GSSG)glutathione GSSG/GSHQuantificationKit 7
2. DNA Damage Detection QuantificationofdamagedbaseingenomicDNA DNADamageQuantificationKit-APSiteCounting- 8
NitratedbaseofDNAandRNAdetection Anti-Nitroguanosineantibodies 11
Standardagentof8-nitroguaninedetection 8-Nitroguanine(Iyophilized) 12
3. Lipid Peroxide Detection Detectionoflipidperoxidebyfluorescent microscopyandflowcytometry
Liperfluo 13
DetectionoflipidperoxidebyHPLC DPPP 14
4. Radical Detection DetectionofproteinorDNAradicalbyWB,ELISA, andimaging
DMPO 15
DetectionofradicalbyEPR DMPO 16
DetectionofsuperoxideradicalbyEPR BMPO 17
5. Nitric Oxide(NO) Detection NOscavenger,NOdetectionbyEPR Carboxy-PTIO 18
DetectionofNO2-byfluorometricanalysis 2,3-Diaminonaphthalene(forNOdetection) 19
DetectionofNObyEPR DTCSNa 20
DetectionofNObyEPR MGD 21
6. Nitric Oxide(NO) Donation NOdonor(stableinacidiccondition) NORs 22
NOdonor(stableinalkalinecondition) NOCs 23
NOdonorfromnitrosothiolcompound S-Nitrosoglutathione 25
ONOO-(peroxynitrite)donor SIN-1 26
7. AGEs Research StandardsubstanceforAGEprecursor 3-Deoxyglucosone 27
Detectionof3-DGbyHPLC(fluorometric) 3-DeoxyglucosoneDetectionReagent 27
Dojindo Oxidative Stress Products
O2
hypoxanthine
Carbamylation
Protein
HCitProteinMPO
Smoking/Diet
Xanthine
XO
O2•-
e-
DMPOBMPO
H2O2•OH H2O
e- e- e-
H2O
O2•- O2
SODSOD Assay Kit-WST
LOH+H2O
GPx
LOOH GSH
GSSG
Total GlutathioneQuantification Kit
GRGSSG/GSH
Quantification Kit
NADP+
NADPH
TR
redTRX
oxTRX
NAD+
NADH
GPx,Cat
HOCl
MPOCl-
O2
MDA,HNEetcLOOH
Carbonyl-protein
DNAdamage(8-OhdG,APsiteetc)
DPPP
DPPP
Liperfluo
Liperfluo
DNA Damage Quantification Kit
sugarlipid
LH
Protein
DNA
Cat:catalaseGPx:glutathioneperoxideGR:glutathionereductaseMPO:myeloperoxideTR:thioredoxinreductaseTRX:thioredoxinLOOH:lipidperoxideXO:xanthineoxidase
hypoxanthine Xanthine
NAD+NADHG6PD
gluconolactone-6-P G6-P
glucose
NAD+NADHAR
sorbitol
fructose-6-phosphate
glyceraldehyde-3-phosphate
1,3-diphosphoglycerate
pyruvicacid
TCAcycle
mitochondrial electron transfer system
fructose
3-DGmethylglyoxalglycolytic
pathway
Amadorirearrangement
NAD+
NADHProtein-NH2
SDH
AGE
O2 ROS Protein
polyol pathway
glycation
O2
ROS
dehydrationcondensation
3-DeoxyglucosoneDetection Reagent
autoxidation
Dojindo Products
Dojindo Products
DMPOBMPO
H2O2RelatedOxidativeStress
DiabetesandOxidativeStress
Oxidative Stress Map
ROS:reactiveoxygenspeciesAR:aldosereductaseSDH:sorbetoldehydrogenaseAGE:advancedglycationendproduct
Dojindo Oxidative Stress Products
NO2-,NO3
-,H+
N2O4N2O3
H2O H2O
•NO2•NO
ONOO-
SIN-1
H+
•NO
R-SNO
R-SS-R
•NO Fe(II)-NO+
Fe(III)
R-SH
R-SH
O21/2
•OH
NO donor(NOCs, NORs)
Carboxy-PTIO
MGD
L-citruline
L-arginine
leukocyte
•NO
O2
ApoptosisDevelopmentReproduction
GrowthControl
iNOSNOSinhibitor
NAD(P)H NAD(P)+
LeukocyteNAD(P)Hoxidase
O2•-
Pathogens
Defencecontrol
resistance artery(artery)
bloodvessel
Hb-O2
bloodflowoxygentransportNO-Hb HbNO3
-
urinecontraction-relaxation
FlowControl
O2L-arginine
L-citruline
eNOS
dietary(extrinsic)Nitrate(NO3
-)
Nitrite(NO2-)
erythrocyte
mammals Nox family oxidasegenelocus(human)
aminoacidnumber(human)
tissueofhighexpression(function)
Nox1 Xq22 564 colonicepithelium,vascularsmoothmuscle(bloodpressureelevationofAngII)
Nox2/gp91phox Xp21.1 570 phagocyte,Blymphocyte(phylaxis)
Nox3 6q25.1-26 568 innerear,fetalkidneys(otolithformation)
Nox4 11q14.2-21 578 renaltublar,vascularendothelial
Nox5 15q22.31 737 spermatozoon,spleen,lymphocyte
Duox1 15q21 1,551 thyroid,bronchial
Duox2 15q21 1,548 thyroid,largeintestine(syntheticthyroidhormone)
NitricOxide(NO)RelatedStress
PropertyofNADPHOxide(NOX)Family
Oxidative Stress Map
Dojindo Products
DTCS Na
Dojindo Oxidative Stress Products
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*SignalingandmetabolicpathwayswereeditedunderthesupervisionofDr.KeizoSato/KyushuUniversityofHealthandWelfare.
VI IV
senescencecarcinogenesisneuropathydiabetesmellitussterilityrheumatismetc.
ADP ATP ATP
ROS+mitochondrialdamage
Citricacidcycle
Acetyl-CoA
NADHNAD+
e-
e-
H2O
*I - V:Complex I - V
CoenzymeQ
IIICytochromeC
H+ H+ H+H+
H+H+
H+
H+
H+O2
O2•- O2
•-
mitochondrion
MitochondrialOxidativeStress
ReactiveOxygenSpeciesRelatedinAtherosclerosis
Oxidative Stress Map
Dojindo Oxidative Stress Products
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Introduction
OxygenisaveryimportantmoleculeforthesynthesisofbiologicallyactivematerialssuchashormonesandATP. Acquisition of the ability to utilize oxygenwas a significant driving force for the evolutionof life.Oxygenactivates various enzymes in cells andactivatedoxygen species are involved in theoperation of cell functions.Although oxygen itself is an essential element of life,molecules in cells,
suchasDNAandproteins,aresometimesdamagedbyreactiveoxygenspecies(ROS)inwhatiscalledoxidativestress.ROScanbecreatedbymetabolism,ionizingradiation,andcarcinogeniccompoundsthatdirectlyinteractwithDNA.Duringmetabolism,asmallportionofoxygenisconvertedtosuperoxideanionbyoneelectronreduction;superoxideanionisthenconvertedtooxygenandhydrogenperoxidebysuperoxidedismutase(SOD).Hydrogenperoxideisreducedtowaterbycatalaseorglutathioneperoxidase.However,ifhydrogenperoxideisnotcompletelyreducedbytheseenzymes, itcangenerateanextremelyreactivehydroxyradicalwhenoxidizedby iron(Fentonreaction).HydroxyradicalisalsogeneratedbyUVirradiationordirectlyfromwaterbyionizingradiation.Hydroxyradicalreactswithlipidtogeneratelipidperoxide.However,notallROSareunwanted.Hypochloriteion,anROSderivedfromhydrogenperoxidebymyeloperoxidaseinneutrophils,hasgermicidalactivity.Nitricoxide,alsoknownasendothelial-derivedrelaxationfactor,isgeneratedbyNOsynthetase.However,NOandsuperoxideanionmayreacttogenerateperoxynitrite,whichiscytotoxic.
TheROSandreactivenitrogencompoundshavemanydifferentactivitiesinbiologicalsystems.Inresponse,aerobicorganismscreateddefensemechanismstoavoidoxidativestress.Oxidativestresshasrecentlybecomethefocusofmanystudiesseekingtounderstandthesedefensemechanismsandtherelationshipsbetweenoxidativedamageanddiseaseoragingprocesses.Tothisend,manyassaymethodshavebeendevelopedforthedetectionofROS-relatedorROS-derivedsubstancessuchassuperoxideanion,superoxidedismutase,glutathione,glutathionereductase,glutathioneperoxidase,DNAlesions,8-oxoguanine,8-nitroguanosine,andproteincarbonyl.
Nitricoxide(NO)hasbeenidentifiedasanendothelial-derivedrelaxationfactorandantiplateletsubstance.Itservesasaneurotransmitterwhenderivedfromaneutrophil,andasacytotoxicsubstancewhenderivedfromanactivatedmacrophage.NOreactswithsuperoxideaniontogeneratehighlytoxicperoxynitrite.ThereactionrateofNOwithsuperoxideisthreetimesthatofSOD.Insomecases,NOalsoactivatescyclooxygenase.ThemostimportantroleofNO is thought tobe theactivationofguanylatecyclase.Recently,publishedNOresearchhas reportedmanycontradictoryresults,whichareduetoNO’suniquechemicalproperties.SinceNOisafreeradical,itisveryreactiveandunstable.NOchangesitsforminacomplexmannerimmediatelyafterappearinginabiologicalenvironment.EachofNO’smetabolitesmighthavedifferentbioactivitiesfromNOitself.Forthisreason,it isvitaltoseparatelyinvestigateeachfunctionoftheNO-relatedmetabolites.
Dojindo Oxidative Stress Products
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Measurementofsuperoxidedismutaseactivity
SOD Assay Kit-WST
Superoxidedismutase(SOD),whichcatalyzesthedismutationofthesuperoxidean-ion(O2
.-)intohydrogenperoxideandmolecularoxygen,isoneofthemostimpor-tantantioxidativeenzymes.InordertodeterminetheSODactivity,severaldirectandindirectmethodshavebeendeveloped.Amongthesemethods,anindirectmethodus-ingnitrobluetetrazolium(NBT)iscommonlyusedduetoitsconvenienceandeaseofuse.However,thereareseveraldisadvantagestotheNBTmethod,suchaspoorwa-tersolubilityoftheformazandye,andtheinteractionwiththereducedformofxanthineoxidase.SODAssayKit-WSTallowsaveryconvenientandhighlysensitiveSODassaybyuti-lizingDojindo’shighlywater-soluble tetrazoliumsalt,WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium,monosodiumsalt),whichproducesawater-solubleformazandyeuponreductionwithasuperoxideanion(Fig.1).TherateofWST-1reductionbysuperoxideanionislinearlyrelatedtothexanthineoxidaseactivityandisinhibitedbySOD(seefigurebelow).Therefore,theIC50(50%inhibitionconcentra-tion)ofSODorSOD-likematerialscanbedeterminedusingcolorimetricmethods.
1. Preparation of Sample Solutions► ErythrocytesorPlasma1. Centrifuge2-3mlofanticoagulant-treatedblood(suchasheparin10U/mlwith
finalconcentration)at600xgfor10minutesat4°C.2. Removethesupernatantanddiluteitwithsalinetouseasaplasmasample.
Addsalinetothepellettopreparethesamevolume,andsuspendthepellet.3. Centrifugethepelletsuspensionat600xgfor10minutesat4°C,anddiscard
thesupernatant.4. Addthesamevolumeofsaline,andrepeatStep3twice.5. Suspendthepelletwith4mldistilledwater,thenadd1mlethanoland0.6ml
chloroform.6. Shakethemixturevigorouslywithashakerfor15minutesat4°C.7. Centrifugethemixtureat600xgfor10minutesat4°Candtransfertheupper
water-ethanolphasetoanewtube.8. Mix0.1mloftheupperphasewith0.7mlofdistilledwater,anddilutewith0.25%
ethanoltopreparesamplesolution.
► Tissue(100mg)1. Wash the tissuewith saline to removeasmuchbloodaspossible.Blot the
tissuewithpapertowelsandthenmeasureitsweight.2. Add400-900µl sucrosebuffer (0.25Msucrose, 10mMTris, 1mMEDTA,
pH7.4)andhomogenizethesampleusingTeflonhomogenizer.Ifnecessary,sonicate thehomogenized sampleonan icebath (60Wwith0.5 secondintervalsfor15minutes).
3. Centrifugethehomogenizedsampleat10,000xgfor60minutesat4°C,andtransferthesupernatanttoanewtube.
4. Dilutethesupernatantwithdistilledwatertopreparesamplesolution.
2. Preparations of Solutions (for one 96-well plate)► WSTworkingsolution
Dilute1mlofWSTSolutionwith19mlofBufferSolution► Enzymeworkingsolution
CentrifugetheEnzymeSolutiontubefor5seconds.Mixbypipettinganddilute15μlofEnzymeSolutionwith2.5mlofDilutionBuffer.
► SamplesolutionDilutesamplesolutionpreparedwithDilutionBufferorSaline.e.g.)dilutionrate:x1(nodilution),x1/5,x1/52,x1/53,x1/54,x1/55,x1/56
Contents of the Kit 500testsWSTSolution 5mlx1EnzymeSolution 100μlx1BufferSolution 100mlx1DilutionBuffer 50mlx1
Required Equipment & MaterialsMicroplateReader(450nmfilter)96-wellmicroplate2-20μl&20-200μlmulti-channelpipettesIncubator(37°C)
Fig.1 SOD inhibition assay mechanism
Fig. 2 Absorption spectrum of WST-1 formazan
Fig. 3 Serial dilution process
Product Code: S311
Fig. 4 Assay procedure
4)
xanthineoxidase
Samplesolution
50μl
50μl50μl 50μl 50μl 50μl
Dilutionbuffer200μl
Dilutionbuffer200μl
Dilutionbuffer200μl
Dilutionbuffer200μl
Dilutionbuffer200μl
Dilutionbuffer200μl
1/51/521/531/541/551/56
1) 2)
3)
300400500600700Wavelength/nm
2.0
1.0
0
Absorbance
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch
Dojindo Oxidative Stress Products
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3. General Protocol (refer to Table 1, Fig. 4 and Fig. 5)1. Add20μlofsamplesolutiontoeachsamplewellandblank2well,andadd20
μlofddH2O(double-distilledwater)toeachblank1andblank3well.2. Add200μlofWSTWorkingSolutiontoeachwell,andmixbypipetting.3. Add20μlofDilutionBuffertoeachblank2andblank3well.4. Add20μlofEnzymeWorkingSolutiontoeachsampleandblank1well.5. Incubatetheplateat37ºCfor20minutes.6. Readtheabsorbanceat450nmusingamicroplatereader.7. CalculatetheSODactivity(inhibitionrate%)usingthefollowingequation.
SODactivity(inhibitionrate%)={[(Ablank1-Ablank3)-(Asample-Ablank2)]/(Ablank1-Ablank3)}x100
4. Inhibition CurveAsFig.6shows,SODAssayKit-WSTcanmeasure100%inhibitionbecauseWST-1doesnotreactwiththereducedformofxanthineoxidase(XO).
5. Definition of Unit(U)One unit is defined as a point where a 20 μl of sample solution gives 50% inhibition of a colorimetric reaction between WST-1 and superoxide anion.*UnitdefinitiondifferfromtheunitdefinitionofCytochromeCassay.
6. Calculate Unit(U)1. Calculateadilutionratiowheretheinhibitioncurvegives50%inhibition.2. SODunitinoriginalsamplecanbecalculatedbymultiplyingthedilutionrate.
7. Example of Calculating Unit(U): Erythrocytes(x108 dilution sample)1. Calculateadilution ratio from thepointof IC50 in the inhibitioncurve.Fig.7
givesthedilutionrateatIC50of1/1.8.2. Accordingtothedefinitionofunit,20μlofthissampleiscalculated1.8U.3. SODunitper1mlof thissamplesolutioncanbecalculatedby the following
equation,1.8/0.02=90.0U/ml.4. Original erythrocytes samplewasdiluted108 timesduring the sample
preparation.To calculate theSODunit in theoriginal,multiply 90.0U/ml by108.TheSODunitintheoriginalsampleis9,720U/mlofblood.
*SODunitcanbecalculatedasU/gramorU/mg.
8. Distinguish Mn-SOD from Cu/Zn-SOD and EC-SODMn-SODcanbemeasuredbyblocking theCu/Zn-SODandEC-SODactivity usingpotassiumcyanide(KCN)orDiethyldithiocarbamate(DDC).
9. InterferenceReducingagents,suchasascorbicacidsandreducedformsofglutathiones,interferewith theSODassay.Table2 shows the concentrationsofmaterials that cause10%increaseintheO.D.value.Ifsamplecontainsthesematerials,pleasedilutethesampletoavoidtheinterfere.
10. References1. J.M.McCord,et al.,AnEnzymicFunctionforErythrocuprein(hemocuprein).J Biol Chem.1969;244:6049-6055.2. B.L.Geller,et al.,AMethodforDistinguishingCu,Zn-andMn-ContainingSuperoxideDismutases.Anal Biochem.1983;128:86-
92.3. S.Goldstein,et al.,ComparisonBetweenDifferentAssaysforSuperoxideDismutase-likeActivity.Free Rad Res Commun.
1991;12:5-10.4. R.H.Burdon,et al.,ReductionofaTetrazoliumSaltandSuperoxideGenerationinHumanTumorCells(HeLa).Free Rad
Res Commun.1993;18:369-380.5. M.W.Sutherland,et al.,TheTetrazoliumDyesMTSandXTTProvideNewQuantitativeAssaysforSuperoxideandSuperoxide
Dismutase.Free Radic Res.1997;27:283-289.6. H.Ukeda,et al.,Flow-InjectionAssayofSuperoxideDismutaseBasedontheReductionofHighlyWater-SolubleTetrazolium.
Anal Sci.1999;15:353-357.7. H.Ukeda,et al.,SpectrophotometricAssayforSuperoxideDismutaseBasedontheReductionofHighlyWater-solubleTet-
razoliumSaltsbyXanthine-XanthineOxidase.Biosci Biotechnol Biochem.1999;63:485-488.8. H.Ukeda,et al.,SpectrophotometricAssayofSuperoxideAnionFormedinMaillardReactionBasedonHighlyWater-soluble
TetrazoliumSalt.Anal Sci.2002;18:1151-1154.9. N.Tsuji,et al.,EnhancementofTolerance toHeavyMetalsandOxidativeStress inDunaliella TertiolectabyZn-induced
PhytochelatinSynthesis.Biochem Biophys Res Commun.2002;293:653-659.
Fig. 5 Sample and blank arrangement on a 96-well microplate
Fig. 6 Inhibition curve of Cu/Zn-SOD
Fig. 7 Inhibition curve on erythrocytes sample
Table 2 Minimum Concentrations of Interfering Substances
Table 3 Measurement Examples
sample blank1 blank2 blank3SampleSolution 20μl - 20μl -
ddH2O - 20μl - 20μlWSTWorkingSolution 200μl 200μl 200μl 200μl
DilutionBuffer - - 20μl 20μlEnzymeWorkingSolution 20μl 20μl - -
TotalSODerythrocyte 9,720U/mlofbloodserum 355U/mlofblood
heart(rat) 15,712U/g(wet)liver(rat) 142,907U/g(wet)HeLacell 73U/1x107cellsHL60cell 226U/1x108cells
Detergents
SDS 0.05%Tween20 0.5%NP-40 0.5%TritonX-100 0.2%
SolventsEthanol 25%DMSO 5%
Reducingagents
Glutathione,reducedform
1.25mmol/l
Ascorbicacid 0.1mmol/l
OtherEDTA 2mmol/lBSA 1%w/v
Table 1 Solution and buffer volumes in each well
1 2 3 4 5 6 7 8 9 10 11 12
A sample1,1
B sample1,1/5
C sample1,1/52
D sample1,1/53 sample2 sample3 sample4
E sample1,1/54
F sample1,1/55
G sample1,1/56
H blank1 blank2 blank3
1/1041/1031/1021/101Dilutionratio
100
90
80
70
60
50
40
30
20
10
0
Inhibition(%)
▲ ▲ ▲▲
▲
▲
1/1.8
1/1051/1041/1031/1021/101
Dilutionratio
100
90
80
70
60
50
40
30
20
10
0
Inhibition(%)
▲▲ ▲ ▲ ▲▲
▲
▲▲
▲▲
1. A
nti O
xida
nt
Det
ectio
n2.
DN
A D
amag
e D
etec
tion
3. L
ipid
Per
oxid
e D
etec
tion
4. R
adic
al D
etec
tion
6. N
O D
onor
5. N
itric
Oxi
de
Det
ectio
n7.
AG
Es R
esea
rch
Dojindo Oxidative Stress Products
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Title ReferenceAmyotrophicLateralSclerosisModelDerivedfromHumanEmbryonicStemCellsOverexpressingMutantSuperoxideDismutase1
T.Wada,et al.,Stem Cells Trans Med.2012;1:396
Copperchelationbytetrathiomolybdateinhibitslipopolysaccharide-inducedinflammatoryresponsesinvivo
H.Wei,et al., Am J Physiol Heart Circ Physiol.2011;301:H712
ANewEnteralDiet,MHN-02,WhichContainsAbundantAntioxidantsandWheyPeptide,ProtectsAgainstCarbonTetrachloride–InducedHepatitis
T.Takayanagi,et al., JPEN J Parenter Enteral Nutr.2011;35:516
EffectsofdietarysupplementationofmethionineanditshydroxyanalogDL-2-hydroxy-4-methylthiobutanoicacidongrowthperformance,plasmahormonelevels,andtheredoxstatusofbroilerchickensexposedtohightemperatures
H.Willemsen,et al., Poult. Sci.,2011;90:2311
RafKinaseInhibitorProteinRKIPEnhancesSignalingbyGlycogenSynthaseKinase-3β
F.Al-Mulla,et al., Cancer Res.2011;71:1334
TemporalchangesintheexpressionofmRNAofNADPHoxidasesubunitsinrenalepithelialcellsexposedtooxalateorcalciumoxalatecrystals
SaeedR.Khan,et al., Nephrol. Dial. Transplant.2011;26:1778
Profilingofsuperoxidedismutaseisoenzymesincompartmentsofthedevelopingbovineantralfollicles
C.Combelles,et al., Reproduction,2010;139:871
Deletionofnuclearfactor-E2-relatedfactor-2leadstorapidonsetandprogressionofnutritionalsteatohepatitisinmice
H.Sugimoto,et al., Am J Physiol Gastrointest Liver Physiol.2010;298:G283
GalliumDisruptsIronUptakebyIntracellularandExtracellularFrancisellaStrainsandExhibitsTherapeuticEfficacyinaMurinePulmonaryInfectionModel
O.Olakanmi,et al., Antimicrob. Agents Chemother.2010;54:244
Ironrestrictionimprovestype2diabetesmellitusinOtsukaLong-EvansTokushimafattyrats
Y.Minamiyama, et al., Am J Physiol Endocrinol Metab,2010;298:E1140
12. FAQ►WhatisthedefinitionofaUnit?
Oneunitisdefinedasapointwhereasamplegives50%inhibitionofacolorimetricreactionbetweenreactivedye(suchascytochromeC,WST-1,nitro-tetrazoliumblueorXTT)andsuperoxideanion.Forexample,iftheO.D.of"Blank1"thatdoesnotcontainanySODis1.0,thesamplethatgives0.5O.D.isdefinedashaving1unitofSODactivity.YoucanusethisunittodeterminetheSODactivityofyoursample.Therefore,SODactivitiesdeterminedusingdifferentdyesormethodsarenotcomparablewitheachother.
►CanIusestandardSODtodetermineSODactivityinsamplesolutions?Yes,youcan.Prepareainhibitioncurve(typicalinhibitioncurve,anddetermineSODactivityinthesamplesolution.SODbovineerythrocytes(CAS#9054-89-1,EC1.15.1.1)canbepurchasedfromSigma(catalog#S7571).
►CanIuseakineticmethodtodetermineSODactivity?Yes,youcanuseakineticmethodforSODassay.Sincetherateofthecolordevelopmentremainsthesameforupto20minutes,measuretheslopefor5minutesduringthislinearphase.
►Thesamplehascolor.CanIstillusethissample?Yes,youcanstillusethesample.Dilutingthesamplewillminimizetheinterference.SubtracttheO.D.ofblank2fromtheO.D.of thesampletocancelout thebackgroundcolor.However, if theSODactivity in thesample is low, itmaynotbemeasurable.
►HowdoIpreparemoreDilutionBuffer?DilutionBuffer isPBS.PleasepreparetheDilutionbufferwithfollowingconcentrations;137mMNaCl,2.7mMKCl,1.47mMKH2PO4,8.1mMNa2HPO4,pH7.4.
►CanIdetermineMn-SODandCu/Zn-SODindependentlyusingthiskit?Yes.InordertomeasureMn-SODactivity,itisnecessarytoblocktheCu/Zn-SODactivityusingpotassiumcyanide(KCN).Adding1mMKCNtosamplescanblockCu/Zn-SODactivitycompletely.TomeasureCu/Zn-SODactivity,measurethetotalSODactivitywithandwithoutKCN,andthensubtracttheMn-SODactivityfromtotalSODactivity.
►HowlongcanIstorethesample?Asamplestoredinafreezerat-80ºCisstablefor1month.
►CanImeasurethelevelsofsuperoxideanionusingthiskit?No.However,youcouldsimplyuseWST-1, insteadof thiskit, tomeasuresuperoxide.Youwouldneedastandard todeterminetheamountofsuperoxideinsamplesolution.Sincesuperoxideisnotstableandreactswithvariousmaterials,itmightbedifficult todeterminethetotalamountofsuperoxidegenerated in thesystem.Thexanthine-xanthineoxidasesysteminthiskitcanbeusedasastandardformeasuringtherelativeamountofsuperoxideproductionineachsample.
11. Recent Publications
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch
Dojindo Oxidative Stress Products
4
Glutathione(GSH) is themostabundant thiolcompound inanimal tissues,planttissues,bacteria,andyeast.GSHhasmanydifferentroles, includingprotection
againstreactiveoxygenspeciesandthemaintenanceofproteinthiolgroups.Duringtheseprocesses,GSHisconvertedintoitsoxidizedform,glutathionedisulfide(GSSG).SinceGSSG is thenenzymatically reducedbyglutathione reductase,GSH is thedominantforminorganisms.DTNB(5,5’-Dithiobis(2-nitrobenzoicacid)),knownasEllman’sReagent,wasdevelopedtodetectthiolcompounds.In1985,Dr.M.E.AndersonsuggestedthattheglutathionerecyclingsysteminvolvingDTNBandglutathionereductasecouldbeusedasahighlysensitiveglutathionedetectionmethod.DTNBandGSHreact togenerate2-nitro-5-thiobenzoicacidandGSSG(Fig.1).Since2-nitro-5-thiobenzoicacid isyellow, theGSHconcentration inasamplesolutioncanbedeterminedbyO.D.measurementat412nmabsorbance(Fig.2).GSHisregeneratedfromGSSGbyglutathionereductaseandwillagainreactwithDTNBtoproduce2-nitro-5-thiobenzoicacid.Thisrecyclingreactionimprovesthesensitivityoftotalglutathionedetection.
1. Preparation of Sample Solutions► Cell(adhesivecell:5x105cells,leukocytecell:1x106cells)1.Collectcellsbycentrifugationat200g for10minutesat4°C.Discard thesupernatant.
2.Washthecellswith300μlPBSandcentrifugeat200gfor10minutesat4°C.Discardthesupernatant.
3.Add80μl10mMHCl,andlysethecellsbyfreezingandthawingtwice.4.Add20μl5%SSAandcentrifugeat8,000gfor10minutes.5.Transfer thesupernatant toanewtube,anduse it for theassay. If the finalconcentrationofSSAisover1%,addddH2OtoreducetheconcentrationofSSAfrom0.5to1%.
► Tissue(100mg)1.Homogenizethetissuein0.5-1.0ml5%SSA.2.Centrifugethehomogenizedtissuesampleat8,000gfor10minutes.3.Transfer the supernatant to a new tube and add ddH2O to reduce theconcentrationofSSAfrom0.5to1%.Useitfortheassay.
► Plasma1.Centrifugeanticoagulant-treatedbloodat1,000gfor10minutesat4°C.2.Transferthetopplasmalayertoanewtubeandadd5%SSAequivalenttohalfofthevolumeoftheplasma.
3.Centrifugeat8,000gfor10minutesat4°C4.Transfer the supernatant to a new tube, and add ddH2O to reduce theconcentrationofSSAfrom0.5to1%.Useitfortheassay.
► Erythrocyte1.Centrifugeanticoagulant-treatedbloodat1,000gfor10minutesat4°C.2.Discardthesupernatantandthewhitebuffylayer.3.Lyse theerythrocyteswith5%SSAequivalent to4 times thevolumeof theerythrocytes.
4.Centrifugeat8,000gfor10minutesat4°C.5.Transfer the supernatant to a new tube, and add ddH2O to reduce theconcentrationofSSAfrom0.5to1%.Useitfortheassay.Erythrocytescanbeisolatedfromtheremainingsamplesolutionaftertheplasmasampleisolation.
*Preparationof5%5-SulfosalicylicAcid(SSA)SolutionNote:SSAisnotincludedinthiskit.1.Dissolve1gSSAin19mlwater.2.Storethesolutionat4°C(stablefor6monthsat4°C).
Fig.1 Principal of total glutathione detection
Fig. 2 Absorption spectrum of 5-Mercapto-2-nitrobenzoic acid
Quantificationoftotalglutathione
Total Glutathione Quantification Kit Product Code: T419
Contents of the KitSubstrate(DTNB) 2vialsEnzymeSolution 50μlx1Coenzyme(lyophilized) 2vialsStandardGSH(lyophilized) 1vialBufferSolution 50mlx1
Required Equipment & MaterialsMicroplateReader(405or415nmfilter)96-wellmicroplate20-200μlmulti-channelpipettesIncubator(37°C)5-sulfosalicylicacid(SSA)
300400500600Wavelength/nm
1.5
1.0
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2. Preparations of Solutions► Substrateworkingsolution
Add1.2mlofBufferSolutiontoonevialofSubstrate,anddissolve.Storethesolutionat-20°C(stablefor2months).
► EnzymeworkingsolutionMixEnzymeSolutionusingpipette.Takeout20µlofEnzymeSolution,andmixitwith4mlofBufferSolution.Storethesolutionat4°C(stablefor2months).
► CoenzymeworkingsolutionAdd1.2mlofddH2OtotheCoenzymevialanddissolve.TheCoenzymevialisdecompressed.UseasyringetoaddddH2O,thenopenthevial.Storethesolutionat-20°C(stablefor2months).
► GSHstandardsolutionAdd2mlof0.5%SSAtoStandardGSHvial,anddissolvetoprepare200µMofGSHstandardsolution.TheStandardGSHvialisdecompressed.Useasyringetoadd0.5%SSA,thenopenthevial.Storethesolutionat-20°C(stablefor2months).Dilute100µlof200µMGSHstandardsolutionbyserialdilutionwith100ulof0.5%SSAinplastictubesasindicatedFig.4.
3. General Protocol (refer to Fig. 3)1.Add20µlofEnzymeworkingsolution,20µlofCoenzymeworkingsolutionand
120µlofBufferSolutiontoeachwell.2. Incubatetheplateat37°Cfor5minutes.3.Add20µlofGSHstandardsolutionand20µlofsamplesolutiontoeachwell.4. Incubatetheplateat37°Cfor10minutes.5.Add20µl ofSubstrateworking solution, and incubate theplate at room
temperaturefor10minutes.6.Readtheabsorbanceat405nmor415nmusingamicroplatereader.7.DeterminconcentrationsofGSHinthesamplesolutionsusingacalibrationcurve.
Sincethecolorimetricreaction isstableandtheO.D. increases linearlyover30min.AtimecourseofthecolorimetricreactionisshownFig.5.Typicalcalibrationcurvespreparedusingthepseudo-endpointmethodisindicatedinFig.6.
4. Calculation of total glutathione (GSH and GSSG) concentrationDeterminethetotalglutathioneconcentrationa)inasamplesolutionusingthefollowingequations.► pseudo-endpointmethod
Total glutathione (GSH+GSSG)=(O.D.sample - O.D.blank)/slopeb)
► kineticmethodTotal glutathione (GSH+GSSG)=(Slopec)
sample - Slopec)blank)/slopeb)
a) Sincethevaluesobtainedbytheseequationsaretheamountof totalglutathionein treated sample solutions, further calculationsarenecessary if theactualconcentrationsofglutathioneinsamplesneedtobedetermined.
b) slopeofthecalibrationcurvec) slopeofthekineticreaction
5. InterferenceReducingagents suchasascorbic acid,β-mercaptoethanol, dithiothreitol (DTT)andcysteine, or thiol reactive compounds suchasmaleimide compounds, interferewiththeglutathioneassay. Therefore,SHcompounds, reducingagentsandSH reactivematerialsshouldbeavoidedduringthesamplepreparation.
6. Notes• Thekitisstablefor6monthsat0to5°C.• Usethereagentsinthekitafterthereagentstemperatureareequilibratedtotheroomtemperature.
• Triplicatemeasurementspersampleisrecommendedtoobtainaccuratedata.• SincethecolorimetricreactionstartsimmediatelyaftertheadditionofSubstrateworkingsolutiontoawell,useamultichannelpipettetoavoidthereactiontimelagofeachwell.
• If theconcentration rangeof totalglutathione inasample isunknown,preparemulti-dilutedsamplesolutions.
• This kit is not forGSSG/GSH ratio determination. ForGSSG/GSH ratiodetermination,pleaserefertoGSSG/GSHQuantificationKit.
7. References1. G.L.Ellman,Tissuesulfhydrylgroups.Arch Biochem Biophys.1959;82:70-77.2. O.W.Griffith,DeterminationofGlutathioneandGlutathioneDisulfideUsingGlutathioneReductaseand2-Vinylpyridine.Anal
Biochem.1980;106:207-212.3. M.E.Anderson,DeterminationofGlutathioneandGlutathioneDisulfideinBiologicalSamples.Methods Enzymol.1985;113:548-555.4. M.A.Baker,et al.,MicrotiterPlateAssayfortheMeasurementofGlutathioneandGlutathioneDisulfideinLargeNumbersof
BiologicalSamples.Anal Biochem.1990;190:360-365.
Fig. 3 Assay procedure
Fig. 5 Time-dependent Abs. increase on eight diferent samples
Fig. 6 Calibration curve prepared using pseudo-endpoint method(10 min
incubation at room temperature)
Fig. 4 Serial dilution process
4)3)
200μMGSH100μl
100μl100μl 100μl 100μl 100μl
0.5%SSA100μl
0.5%SSA100μl
0.5%SSA100μl
0.5%SSA100μl
0.5%SSA100μl
0.5%SSA100μl
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Title ReferenceCopperChaperone-Dependentand-IndependentActivationofThreeCopper-ZincSuperoxideDismutaseHomologsLocalizedinDifferentCellularCompartmentsinArabidopsis
C.Huang,et al. Plant Physiology.2012;158:737-746
IRE1αactivationprotectsmiceagainstacetaminophen-inducedhepatotoxicity K.Yeon,et al., J. Exp. Med.2012;209:307-318
TheNitricOxideProdrugJS-KIsEffectiveagainstNon–Small-CellLungCancerCellsInVitroandInVivo:InvolvementofReactiveOxygenSpecies
A..Maciag,et al., J. Pharmacol. Exp. Ther.,2011;336:313-320
MutationsinPNKDcausingparoxysmaldyskinesiaaltersproteincleavageandstability
Y.Shen,et al., Hum. Mol. Genet.,2011;20:2322-2332
Phosphate-activatedglutaminase(GLS2),ap53-inducibleregulatorofglutaminemetabolismandreactiveoxygenspecies
S.Suzuki,et al., PNAS,2010;107:7461-7466
OxidativeStressandSodiumMethyldithiocarbamate–InducedModulationoftheMacrophageResponsetoLipopolysaccharideInVivo
S.Pruett,et al., Toxicol. Sci.2009;109:237-246
Nrf2EnhancesCellProliferationandResistancetoAnticancerDrugsinHumanLungCancer
S.Homma,et al., Clin. Cancer Res.2009;15:3423-3432
InhibitionofhepaticNiemann-PickC1-like1improveshepaticinsulinresistance K.Irie,et al., Journal of Dental Research.2008;87:456-460
3-Morpholinopropylisothiocyanateisanovelsyntheticisothiocyanatethatstronglyinducestheantioxidantresponseelement-dependentNrf2-mediateddetoxifying/antioxidantenzymesinvitroandinvivo
Y.Keum,et al., Carcinogenesis.2008;29:594-599
CellsDeficientintheFANC/BRCAPathwayAreHypersensitivetoPlasmaLevelsofFormaldehyde
J.Ridpath,et al., Cancer Res.2007;67:11117-11122
9. FAQ►DoIhavetodilutethesamplesolutionpriortotheassay?
Ifyoudonotknowthetotalglutathionelevelofyoursample,multipledilutionsmaybenecessary. If thetotalglutathionelevelofyoursampleislessthan100μM,nodilutionisnecessary.
►Whatinterfereswiththeassay?Reducingagents(suchasascorbicacid,beta-mercaptoethanol,dithiothreitol,andcysteine)andthiolreactivecompounds(suchasmaleimides)interferewiththeglutathioneassay.Therefore,reducingagentsandthiolreactivecompoundsshouldbeavoidedduringthesamplepreparation.
8. Recent Publications
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DistinguishMeasurementofGlutathione
GSSG/GSH Quantification Kit Product Code: G257
Glutathione(γ-L-glutamyl-L-cysteinylglycine)isatripeptidepresentinthebody,andit is involved inantioxidation, drugmetabolism,andother asenzymesubstrate
of glutathioneperoxidase, glutathioneS-transferase, and thiol transferase, etc.Glutathioneisusuallypresentasreducedform(GSH),butGSHisconvertedinto itsoxidizedform(GSSG)bystimulationsuchasoxidativestress.Therefore,theratioofGSHandGSSGhasbeennotedasindexofoxidativestress.TheGSSG/GSHQuantificationkitcontainsMaskingReagentofGSH.TheGSHcanbedeactivated in the samplebyadding theMaskingReagent.Therefore, only theGSSGisdetectedbymeasuringtheabsorption(λmax=412nm)ofDTNB(5,5’-dithiobis(2-nitrobenzoic acid) using theenzymatic recycling system.Also,GSHcanbedeterminedthequantitybysubtractingGSSGfromthetotalamountofglutathione.The kit canbe limited to quantifyGSH/GSSGconcentration from0.5µmol/l to 50µmol/landGSSGconcentrationfrom0.5µmol/lto25µmol/l.
Contents of the KitEnzymeSolution 50μlx1Coenzyme 2vialsBufferSolution 60mlx1Substrate(DTNB) 4vialsStandardGSH 1vialStandardGSSG 1vialMaskingReagent 20μlx1
Required Equipment & MaterialsMicroplateReader(405or415nmfilter)96-wellmicroplate20-200μlmulti-channelpipettesIncubator(37°C)5-sulfosalicylicacid(SSA)Ethanol
Glutathionereductase
GSH
GSSG 5-Mercapto-2-nitrobenzoicacid
(λmax=412nm)
GSSGGSHGSHMasking
GSSGMeasurement
Maskingreagent+Sample
DTNBFig.1 Principal of GSSG/GSH detection
1. General Protocol► PreparationofSampleSolution
PleaserefertoTotalGlutathioneQuantification,page5.► DeterminationofGSSGconcentration1. Add4µlofMaskingSolutiontosamplesolutionand200µlofGSSGstandard
solutiondilutedwith0.5%SSArespectively,thentransfer40µlofthesolutiontoeachwell.
2. Add120µlofBufferSolutiontoeachwellandincubatefor1hourat37°C.3. Add20µlofSubstrateworkingsolutiontoeachwell,thenadd20µlofCoen-
zymeworkingsolutionandEnzymeworkingsolutiontoeachwellrespectively.4. Incubatefor10minutesat37°Candreadtheabsorbanceat405or415nmus-
ingamicroplatereader.5. DetermineconcentrationsofGSSGinthesamplesolutionusingaGSSGcali-
brationcurve(Fig.2).
► Determinationoftotalglutathioneconcentration1. Addsamplesolutionand40µlofGSHstandardsolutiondilutedwith0.5%
SSAtoeachwell.2. Add120µlofBuffersolutiontoeachwellandincubatefor1hourat37°C.3. Add20µlofSubstrateworkingsolutiontoeachwell,thenadd20µlofCoen-
zymeworkingsolutionandEnzymeworkingsolutiontoeachwellrespectively.4. Incubatefor10minutesat37°Candreadtheabsorbanceat405or415nmus-
ingamicroplatereader.5. Determineconcentrationsof totalglutathione in thesamplesolutionusinga
GSHcalibrationcurve(Fig.3).
► CalculatingtheconcentrationofGSHGSH(conc.)=totalGlutathione(conc.)-2xGSSG(conc.)
2. Recent Publications
Fig. 2 Determination of the concentration of GSSG
Fig. 3 Determination of the concentration of total glutathione
0510152025ConcentrationofGSSG(μmol/l)
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Title Reference
DiurnalVariationofcadmium-inducedmortalityinmice N.Miura,andT.Hasegawa,et al., J. Toxicol. Sci.2012;37:191
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1. Purification of genomic DNASeveraldifferentmethodsandproductsareavailablefortheisolationofgenomicDNAfromsamplessuchasmembranebindingmethod,guanidine/detergentlysismethod,andpolyelectrolyteprecipitationmethod.Amongthesemethods,theguanidine/detergentlysismethodissimple,anditgiveshighlypurifiedgenomicDNAfortheARP-basedabasicsitesdetection.Duringthepurificationprocess,avoidheatingoftheDNAsolution.DeterminetheconcentrationandpurityofthepurifiedgenomicDNAusingthespectrophotometer*andagarosegelelectrophoresis.DissolvethegenomicDNAinTEattheconcentrationof100μg/ml.ItisimportantforanaccurateassaythattheDNAconcentrationisadjustedexactlyto100μg/ml.
*1OD260nm=50μg/ml.TheratioofOD260nm/OD280nmofhighlypurifiedDNAsolutionis1.8orhigher.Proteincontaminationinthesamplesolutionmaycauseapositiveerror.
2. General Protocol► ARPreaction1. Mix10µlofpurifiedgenomicDNAsolution(100µg/ml)and10µlofARP
Solutionina0.5mltube,andincubateat37°Cfor1hour.2. WashtheinsideoftheFiltrationTubewith100µlofTEtwice.3. Add380µlofTEtothereactionsolution,andtransferthesolutiontothe
FiltrationTube.4. CentrifugetheFiltrationTubeat2,500xgfor15minutes,anddiscardthe
filtratesolution.5. Add400µlofTEtotheFiltrationTubeandresuspendtheDNAonthefilter
withapipette.6. CentrifugetheFiltrationTubeat2,500xgfor15minutes.a)7. Add200µlofTEtotheFiltrationTubetoresuspendtheDNAonthefilterwith
apipette.8. TransfertheDNAsolutiontothe1.5mltube,andadd200µlofTEagainto
theFiltrationTubetotransfertheARP-labeledDNAonthefiltercompletelytothe1.5mltube.b)
9. StoretheARP-labeledgenomicDNAsolutionat0to5°C.a) IftheDNAsolutionstillremainsonthefilterafterthecentrifugation,spinforanother
5minutes.b) RecoveryrateofDNAusingthefiltrationtubeis90%,sotheconcentrationofthe
ARP-labeledDNAis2.25µg/ml.FormoreaccuratedeterminationofthenumberofabasicsitesinthesampleDNA,werecommendmeasuringtheDNAconcentration.
Contents of the Kit5samplesARPSolution(10mMARP) 100μlx1ARP-DNAStandardSoln.* 250μlea.(0,2.5,5,10,20,40APsites/100,000bp)DNABindingSolution 10mlx1SubstrateSolution 10mlx1TEBuffer 15mlx1HRP-Streptavidin 25μlx1WashingBuffer 1packFiltrationTube 5tubes96-wellMicroplate/Ubottom 1plate
20samplesARPSolution(10mMARP) 250μlx1ARP-DNAStandardSoln.* 250μlea.(0,2.5,5,10,20,40APsites/100,000bp)DNABindingSolution 10mlx1SubstrateSolution 10mlx1TEBuffer 40mlx1HRP-Streptavidin 25μlx1WashingBuffer 1packFiltrationTube 20tubes96-wellMicroplate/Ubottom 1plate
Required Equipment & MaterialsMicroplateReader(650nmfilter)10μl,100-200μland1mlpipettes50-250μlmulti-channelpipettesIncubator(37°C)0.5mland1.5mltubeCentrifugePaperTowel
Fig. 1 Reaction of AP site with ARP
QuantificationofdamagedbaseingenomicDNA
DNA Damage Quantification Kit-AP Site Counting- Product Code: DK02
OxidativedamagetoDNAisaresultofitsinteractionwithreactiveoxygenspecies(ROS), in particular, thehydroxy radical.Hydroxy radicals,whichareproduced
fromsuperoxideanionandhydrogenperoxidebytheFentonreaction,producemultiplemodificationsinDNA.OxidativeattacksbyhydroxyradicalsonthedeoxyribosemoietywillleadtothereleaseoffreebasesfromDNA,generatingstrandbreakswithvarioussugarmodificationsandsimpleabasicsites(APsites).Infact,APsitesareoneofthemajortypesofDNAdamagegeneratedbyROS.AldehydeReactiveProbe (ARP;N’-aminooxymethylcarbonylhydrazin-D-biotin) reactsspecificallywithanaldehydegrouppresentontheopenringformoftheAPsites(Fig.1).ThisreactionmakesitpossibletodetectDNAmodificationsthatresultintheformationofanaldehydegroup.AftertreatmentwithexcessARPreagent,alloftheAPsitesonDNAaretaggedwithabiotinresidue.Thesebiotin-taggedAPsitescanbequantifiedusingtheavidin-biotinassay,followedbycolorimetricdetectionwitheitherperoxidaseor alkalinephosphatase conjugated to theavidin.DNADamageQuantificationKitcontainsallthenecessarysolutionsfordetectingbetween1to40APsitesper1x105basepairs.
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► DeterminationofthenumberofAPsiteinDNADay1
1. Dilute90µloftheARP-labeledgenomicDNAwith310µlofTE.2. Add60µlofARP-DNAStandardSolutionperwell.Usethreewellsper1
standardsolution.3. Add60µlofthedilutedARP-labeledgenomicDNAsolutionperwell.Useat
leastthreewellsper1sample.4. Add100µloftheDNABindingSolutiontoeachwell,thenallowtheplateto
remainatroomtemperatureovernight.Day2
1. Preparestocksolutions» WashingBuffer:DissolvethecontentsoftheWashingBufferpacketin
1Lofdeionizedordistilledwater.StorethisWashingBuffersolutionatroomtemperature.
» HRP-Streptavidinsolution:DiluteHRP-StreptavidinwithWashingBuffertoprepare1/4000dilutedworkingsolution.*
» 1/4000dilutionpreparation:CentrifugeHRP-Streptavidintubefor30seconds.Add10µlofHRP-Streptavidininto40mlofWashingBuffersolution,andmixwell.
*Sincethisworkingsolutionisnotstable,alwaysusefreshlypreparedsolution.
2. DiscardtheDNABindingSolutioninthewells,andwashthewellwith250µlWashingBuffer5times.
3. Add150µlofdilutedHRP-Streptavidinsolutiontoeachwell,andincubatetheplateat37°Cfor1hour.
4. Discardthesolutioninthewell,andwashthewellwith250µlWashingBuffer5times.b)
5. Add100µlofSubstrateSolutiontoeachwell,andincubateat37°Cfor1hour.6. MeasuretheO.D.at650nmwithin1houraftertheincubationisfinished,and
prepareacalibrationcurveusingthedataobtainedwithARP-DNAStandardsolutions.
7. DeterminethenumberofabasicsitesinthegenomicDNAusingthecalibrationcurve.
3. Notes1. Pleasestorethekitat0-5°C.Donotfreeze.StoreWashingBuffersolutionat
roomtemperature.2. AP-DNAisnotstable.PleasetreatitwithARPandpurifywithFiltrationTube
aftertheisolationofgenomicDNAfromasample.3. PurifiedARP-DNAsolutioninTEbufferisstableoveroneyearat0-5°C
storage.4. AfterthespinningofFiltrationTubeforARP-labeledDNApurification,add200
µlTEimmediately.IftheDNAstaysinFiltrationTubeformorethan30minutesafterthespinning,theDNArecoveryratiomaydecline.
5. γ-Ray-sterilizedtubesmaycauseDNAbindingonthesurfaceofthetubeduringthemixingoftheDNAsolutionwithDNABindingSolution.IfyouprefertomixARP-DNAsolutionwithDNABindingSolutioninatuberatherthanmixingtheminawell,pleaseavoidusingg-ray-sterilizedtubes.
6. Ifthe650nmfilterisnotavailableforthemeasurementofO.D.afterthecolordevelopment,transfer50µlofthesolutionineachwelltoawellofanewplate(notprovided).Then,add50µlof1Msulfuricacid,andmeasuretheO.D.at450nm.
7. Remainingsolutioninawellmaycauseerror,sopleaseremovethesolutionthoroughlybytappingtheplateonapapertowelineachstep.
4. References1. T.Lindahl,et al.,RateofDepurinationofNativeDeoxyribonucleicAcid.Biochemistry.1972;11:3610-3618.2. M.Liuzzi,et al.,ANewApproachtotheStudyoftheBase-excisionRepairPathwayUsingMethoxyamine.J Biol Chem.
1985;260:5252-5258.3. A.Sancar,et al.,DNARepairEnzymes.Annu Rev Biochem.1988;57:29-67.4. M.Weinfeld,et al.,ResponseofPhageT4PolynucleotideKinaseTowardDinucleotidesContainingApurinicSites:Design
ofa32P-postlabelingAssayforApurinicSitesinDNA.Biochemistry.1990;29:1737-1743.5. B.X.Chen,et al.,PropertiesofaMonoclonalAntibodyfortheDetectionofAbasicSites,aCommonDNALesion.Mutat
Res.1992;273:253-261.6. J.A.Gralnick,et al.,TheYggXProteinofSalmonellaentericaIsInvolovedinFe(II)TraffickingandMinimizestheDNA
DamageCausebyHydroxylRadicals:ResidueCYS-7isEssentialforYggXFunction.J Biol Chem.2003;278:20708-20715.
Fig. 2 Assay procedure
Fig. 3 Typical calibration curve of DNA Damage Quantification Kit
1. Anti O
xidant D
etection2. D
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etection4. R
adical Detection
6. NO
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5. Nitric O
xide D
etection7. A
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esearch
010203040NumberofAPsites/100,000bp
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Absorbanceat650nm
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Title Reference
NovelRoleofBaseExcisionRepairinMediatingCisplatinCytotoxicity A.Kothandapani,andS.M.Patrick,et al.,J. Biol. Chem.2011;286:14564-14574
Cadmiumaffectsmetabolicresponsestoprolongedanoxiaandreoxygenationineasternoysters(Crassostreavirginica)
I.O.Kurochkin,andI.M.Sokolova,et al.,Am J Physiol Regulatory Integrative Comp Physiol.2009;297:R1262-R1272
ImpairmentofAPE1FunctionEnhancesCellularSensitivitytoClinicallyRelevantAlkylatorsandAntimetabolites
DanielR.,andD.M.Wilson,et al., IIIMol. Cancer Res.2009;7:897-906
TemporaryPretreatmentWiththeAngiotensinIIType1ReceptorBlocker,Valsartan,PreventsIschemicBrainDamageThroughanIncreaseinCapillaryDensity
Jian-MeiLi,andM.Horiuchi,et al.,Stroke.2008;39:2029-2036
Bcl2InhibitsAbasicSiteRepairbyDown-regulatingAPE1EndonucleaseActivity J.Zhao,andX.Deng,et al., J. Biol. Chem.2008;283:9925-9932
CockaynesyndromeBproteinstimulatesapurinicendonuclease1activityandprotectsagainstagentsthatintroducebaseexcisionrepairintermediates
H.Wong,andD.M.Wilson,et al., IIINucleic Acids Res.2007;35:4103-4113
AngiotensinII-InducedNeuralDifferentiationviaAngiotensinIIType2(AT2)Receptor-MMS2CascadeInvolvingInteractionbetweenAT2Receptor-InteractingProteinandSrcHomology2Domain-ContainingProtein-TyrosinePhosphatase1
Jian-MeiLi,andM.Horiuchi,et al., Mol. Endocrinol.2007;21:499-511
ADominant-NegativeFormoftheMajorHumanAbasicEndonucleaseEnhancesCellularSensitivitytoLaboratoryandClinicalDNA-DamagingAgents
DanielR.McNeillandDavidM.Wilson,IIIMol. Cancer Res.2007;5:61-70
Bcl2SuppressesDNARepairbyEnhancingc-MycTranscriptionalActivity Z.Jin,andX.Deng,et al., J. Biol. Chem.2006;281:14446-14456
FolateDeficiencyIncreasesPostischemicBrainInjury M.Endres,andK.Gertz,et al., Stroke.2005;36:321-325
6. FAQ►CanIusesingle-strandedDNAorRNA?
No,youcannotusethiskit todeterminethenumberofabasicsitesinsingle-strandedDNAorRNA.TheO.D.readingofsingle-strandedDNAwillbenearlytwicethatofdouble-strandedDNAbecauseofthebindingefficiencyonthemicroplate.
►HowshouldgenomicDNAbestored?PrepareaDNApelletandstoreat-20°Cor-80°CiftheDNAcannotbelabeledwithARPimmediatelyafterisolation.AfterARPlabeling,thesamplecanbestoredat4°CinTEBufferforseveralmonths.
►HowshouldIpreparetheDNA?YoucanusegeneralprotocolsorcommerciallyavailableDNAisolationkits.Between2to4abasicsitesper1x105basepairswillbecreatedduringtheDNAisolationprocess.Therefore,usethesameisolationmethodtoprepareeachDNAsample.
►HowcanIdeterminethenumberofabasicsitesiftherearemorethan40per1x105basepairs?Simplydilute theARP-labeledsampleDNAwith0.5μgpermldouble-strandedgenomicDNA,suchascalf thymusorsalmonspermDNA,usingTEBuffer.
►WhatshouldIdoifthesampleDNAconcentrationislessthan100μgperml?YoucaneitheruseafiltrationtubetoconcentrateyoursampleDNAorethanolprecipitationtorecoverDNAasapelletandthenre-dissolveittopreparea100μgpermlsolution.
►WhatshouldIdoifthesampleDNAislessthan1μg?AddthesamevolumeofARPSolutionandfollowthemanual.TherecoveryoftheARP-labeledDNAmaybelowerthantheusualreactions,someasuretheARP-labeledDNAsolution.Theaveragerecoveryrateofthe0.5μgDNAand0.25μgDNAis70%and50%,respectively.
5. Recent Publications
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Fig. 1 Structure of 8-Nitroguanosine
8-Nitroguanine is a nitratedbaseofDNAandRNA. It is formedbyperoxynitrite,which is generated fromnitric oxideand superoxide anion radical. It is known
that a largeamount of nitric oxidemoleculesand superoxideanion, generatedbyinflammation, causesnitrationof guanosine.Since chemicallymodifiednucleotidescausemutationduringDNA replication, 8-Nitroguanine is thought to beamarker ofDNAdamagerelatedtomutationandcancer.Anti-NitroguanosineantibodieshavebeendevelopedjointlybyDr.AkaikeatKumamotoUniversityandDojindoLaboratories.
stronglyreact(10μmol/l)8-NO2-guanosine 8-NO2-guanine 8-NO2-cGMP 8-NO2-Xanthine
slightlyreact(>1mmol/l)8-Br-guanosine 8-Br-guanine 8-Br-cGMP 8-Cl-guanine
noreactionguanosine guanine 8-OH-guaninecytosine xanthine adenineadenosine thymine deoxythymidineuracil uridine 3-NO2-tyrosine
2-NO2-imidazole 8-OH-deoxyguanosineFig. 2 Reactivity of Anti-Nitroguanosine
monoclonal antibodyNO2G52(IC50)
Anti-Nitroguanosinepolyclonal antibodyalso recognizes8-nitroguanineand8-nitroguanosine,but it doesnot cross-reactwithnormal guanosine, guanine,
8-hydroxyguanine,or3-nitrotyrosine.Sincethisantibodywaspreparedusingrabbits,itcanbeusedforimmuno-histostainingofrodenttissuessuchasmiceorrats.
NitratedbaseofDNAandRNAdetection
Anti-Nitroguanosine Antibodies
NitratedbaseofDNAandRNAdetection
Anti-Nitroguanosine monoclonal antibody (Clone# NO2G52) Product Code: AB02
Becauseof its veryhigh specificity,monoclonal antibodyNO2G52 recognizes8-nitroguanineand8-nitroguanosine,but it doesnot cross-reactwithnormal
nucleotidebases, 8-hydroxyguanine8-hydroxydeoxy-guanosine, 3-nitrotyrosine,xanthine,or2-nitroimidazole.The specificity ofNO2G52wasdeterminedby a competitiveELISAusing an8-nitroguanosine-BSA-coatedplate.As shown in the figuresbelow,NO2G52has veryhighaffinity for 8-nitroguanineand8-nitroguanosine, and it slightly cross-reactswith8-bromoguanosine,8-bromoguanine,and8-chloroguanine.
1. Specification► Species:mouse(BALB/c)► Clonality:monoclonal► Isotype:IgG1► Concentration:1mg/mlinPBS,contains0.1%ProClinasapreservative
NitratedbaseofDNAandRNAdetection
Anti-Nitroguanosine polyclonal antibody Product Code: AB01
stronglyreact(10μmol/l)8-NO2-guanosine 8-NO2-guanine
noreactionguanosine guanine 8-OH-guanine
3-NO2-tyrosine
Table 1 Reactivity of monoclonal antibody
Table 2 Reactivity of polyclonal antibody
Fig. 3 Reactivity of Anti-Nitroguanosine polyclonal antibody
10-210-1110102
conc.(μmol/l)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Absorbanceat490nm ● ● ●
●
●
●
●●
▲ ▲ ▲
▲
▲
▲
▲▲
■
■
■
■
■
■■ ■
♦ ♦ ♦♦
♦
♦
♦
♦
○
○ ○ ○ ○
○
○
○
□ □ □ □ □ □ □ □◊
◊ ◊ ◊ ◊ ◊ ◊◊X X X X X X X X* * *
* * * * *
▼
▼
▼
▼▼▼▼▼
■
NO2-Guanine Br-Guanosine
NO2-cGMP Guanosine
NO2-Xanthine Xanthine
NO2-Guanosine cGMP
Br-cGMP Control
●▲
♦○
▼
□◊
*X
10-310-210-1110102103
conc.(μmol/l)
3.0
2.5
2.0
1.5
1.0
0.5
0
Absorbanceat490nm
▲▲▲▲▲
▲▲
▲▲▲▲▲▲
▲▲ ■■■■■■■■■
■■■■■■
●●
●
●
●●●
●●
●
●
●●
●
● NO2-GuanineNO2-GuanosineGuanineGuanosineOH-GuanineNO2-Tyrosine
●▲
■▲
■
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch
Type:IgGUnit:50μg
Type:IgGUnit:50μg
Dojindo Oxidative Stress Products
12
8-Nitroguanine(lyophilized)C5H4N6O3=196.12Unit:100μg
Fig. 4 Tissue staining with Anti-Nitroguanosine antibody
8-Nitroguanine(lyophilized) ismadebythe lyophilizationof itsphosphatebufferedsalinesolution,and isused in immunohistochemistry forabsorption testing.
Adding0.4mlofdistilledwatertothe8-Nitroguaninepowderproducesa1.2mmol/lof8-Nitroguaninesolution.8-Nitroguanine/PBSsolutionisstableforonemonthat4°C.Ifanantibodypre-treatedwithexcessive8-Nitroguanineshowsnegativestaining, thenthesubsequentpositivestainingwiththisantibodywillbespecificfor8-nitroguanineor8-nitroguanosineformedinDNAorRNA.
1. Example of Immunostaining(Fig. 4)► Sample
Influenzavirus-infectedmouselung► Immunostaining1. Fixthemouselungwith2%periodate-lysine-paraformaldehyde.2. Addanti-nitroguanosineantibody(10µg/ml)tothelungsample.3. Addalkalinephosphatase-conjugatedsecondaryantibody.4. StainthesamplewithVectorredsubstratekitI.
2. Notes1. Freezeandthawcyclescancausedegradationoftheantibody.Afteropening,
storeintherefrigerator.2. If8-Nitroguanosinestainingwasobservedwithpolyclonalantibody,itis
reccomendedtoconfirmtheexperimentalverificationasfollows,» nostainingisobservedbythecompetitionwith8-Nitroguaninestandard» nostainingisobservedbytreatingthesamplewithreducingagent,such
asSodiumhydrosulfite3. Monoclonalantibodycanbeusedonhumansamplesduetothehighactivity
andselectivity.
Standardagentof8-Nitroguaninedetection
8-Nitroguanine(lyophilized) Product Code: N455
3. Specification► Species:mouse(BALB/c)► Clonality:monoclonal► Isotype:IgG1► Concentration:1mg/mlinPBS,contains0.1%ProClinasapreservative
4. References1. T.Akaike,et al.,8-nitroguanosineformationinviralpneumoniaanditsimplicationforpathogenesis,PNAS.2003;100:685-
690.2. J.Yoshitake,et al.,NitricoxideasanendogenousmutagenforSendaiviruswithoutantiviralactivity.J Virol.2004;78:8709-
8719.3. T.Sawa,et al.,ProteinS-guanylationbythebiologicalsignal8-nitroguanosine3’,5’-cyclicmonophosphate.Nat Chem Biol.
2007;3:727-735.4. M.H.Zaki,et al.,Cytoprotectivefunctionofhemeoxygenase1inducedbyanitratedcyclicnucleotideformedduring
murinesalmonellosis.J Immunol.2009;182:3746-3756.5. Y.Terasaki,et al.,Guaninenitrationinidiopathicpulmonaryfibrosisanditsimplicationforcarcinogenesis.Am J Respir Crit
Care Med.2006;174:665-673.6. T.Sawa,et al.,Analysisofurinary8-nitroguanine,amarkerofnitrativenucleicaciddamage,byhigh-performanceliquid
chromatography-electrochemicaldetectioncoupledwithimmunoaffinitypurification:associationwithcigarettesmoking.Free Radic Biol Med.2006;40:711-720.
Fig. 5 Detection of guanine nitration on RAW264.7 cell
non-stimulation
LPS + INF-γ
+ L-NMMA
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Liperfluo,aperylenederivative containingoligooxyethylene, isdesignedandexclusivelydevelopedbyDojindoforadetectionoflipidperoxidesandemitsintense
fluorescencebyalipidperoxidespecificoxidationinorganicsolventssuchasethanol.AmongfluorescentprobesthatdetectReactiveOxigenSpecies(ROS),Liperfluoistheonlycompoundthatcanspecificallydetect lipidperoxides.Since theexcitationandemissionwavelengthsoftheoxidizedLiperfluoare524nmand535nm,respectively,bothaphoto-damageagainstasampleandanauto-fluorescencefromthesamplecanbeminimized.Thetetraethyleneglycolgroup linkedtooneendofdiisoquinolineringhelpsitssolubilityanddispersibilitytoaqueousbuffer.ThoughLiperfluooxidizedformisalmostnonfluorescentinanaqueousmedia,itemitsfluorescenceinlipophilicsitessuchasincellmembranes.Thereforeitcaneasilybeappliedtolipidperoxideimagingbyafluorescencemicroscopyandaflowcytometricanalysisforlivingcells.
LiperfluoN-(4-Diphenylphosphinophenyl)-N'-(3,6,9,12-tetraoxatridecyl)perylene-3,4,9,10-tetracarboxydiimideC51H41N2O8P=840.85,Unit:50μgx5
Detectionoflipidperoxidebyfluorescentmicroscopyandflowcytometry
Liperfluo Product Code: L248
Fig. 1 Reaction of Liperfluo with lipid peroxide
80
60
40
20
0
Fluorescenceintensity/A.U
.
400 450 500 550 600 650Wavelength(nm)
Excitation Emission
withoutlipidperoxide
withlipidperoxide
Fig. 3 Reaction selectivity of Liperfluo against the various reactive oxygen species.
15
12
9
6
3
0
F/F 0
1 2 3 4 5 6 7 8
1. Blank2. H2O2
3. •OH4. O2•-
5. NO6. ONOO-
7. ROO•
8. MeLOOH
ROS
Fig. 2 Excitation and emission spectra of Liperfluo with or without lipid per-oxide in ethanol.
1. General Protocol1.Add60μlofDMSOtoavialcontainingLiperfluo(50μg)anddissolvetheproduct(concentration:1mmol/l).-Ifitishardtodissolveitbypipetting,usevortexmixer,sonicatororwarmthesolution.-Coverthesolutionwithanaluminumfoilanduseitwithinadayafterthereconstition.
2.Add10μloftheLiperfluosolutionto1mlofcellculturecontaining1.0x105cells.-HigherfinalconcentrationofDMSOmaycausedamagetocells.-Sincethebackgroundfluorescencemaybeincreasedinculturemedium,replacingthemediumwithanaqueousbuffersuchasPBSisrecommendedbeforetheadditionofLiperfluosolution.
3.Incubatethecellsuspensionat37°Cfor30minutes.4.Analysethecellswithafluorescencemicroscopeoraflowcytometer.-AlthoughtheLiperfluooxidizedformisalmostnon-fluorescentinanaqueoussolution,washthecellswithPBSasnecessaryifthebackgroundfluorescenceishigh.
Ex: 524 nmEm: 535 nm
Liperfluooxidizedformfluorescenceunderlipophiliccondition
2. Live cell imaging of lipid peroxide (Fig. 4)1.InnocurateSH-SY5Ycells(6.0x105cells/well)toa6-wellplate.2.Incubatetheplateat37ºCforovernight.3.AddLiperfluo,DMSOsolution(finalconc.20μM)andincubateat37ºCfor15minutes.
4.AddeitherCumeneHydroperoxide(finalconc.100μM)orAIPH*(finalconc.6mM).5.Incubateat37ºCfor2hours.6.Observefluorescentbymicroscope**(Ex.524nm,Em.535nm).
*AIPH:2,2’-azobis-[2-(2-imidazolin-2-yl)propane]dihydrochloride
**OlympusIX-71epifluorescentmicroscope,mirrorunit:U-MNIBA3,exposuretime:10sec,ISO:800
Fluorescentimage Bright-fieldimage
Fig. 4 Reaction selectivity of Liperfluo against the various reactive oxygen species.
3. References1. K.Yamanaka,et al.,Anovelfluorescentprobewithhighsensitivityandselectivedetectionoflipidhydroperoxidesincells,
RSCAdvances,2012,2,7894.2. N.Soh,et al.,Novelfluorescentprobefordetectinghydroperoxideswithstrongemissioninthevisiblerange.Bioorg Med
Chem Lett.2006;16:2943-2946.3. N.Soh,et al.,Swallow-tailedperylenederivative:anew tool forfluorescent imagingof lipidhydroperoxides.Org Biomol
Chem.2007;5:3762-3768.
4. Specification► Appearance:reddishblackcrystallinepowder► Purity:≥90.0%(HPLC)
DatawaskindlyprovidedfromDr.N.Noguchi,Doshi-shaUniversity,SystemLifeScienceLaboratory.
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch
Dojindo Oxidative Stress Products
14
Lipidperoxidesarederivedfromunsaturated lipids,phospholipids,glycolipidsandcholesterolesters.Infoodindustry,lipidperoxideshavebeenconsideredasoneof
amajorcauseoffooddeterioration.Meanwhile,therearemanyongoingstudiestodaythat investigate themechanismof lipidoxidation inhumandiseases,disorders,andaging.Hence,measuringamountof lipidperoxideinbiologicalsamplesissignificant,andanaccuratemethodfordetectinglowleveloflipidperoxidesiseagerlyanticipated.TBARS(Thiobarbituricacidreactivesubstances)assay isawell-establishedmethodandwidelyusedformeasuring lipidperoxidation.However,MDA(Malondialdehyde),oneofaend-productgeneratedinthelipidperoxidation,isnotreflectedinactuallevelofperoxidationbecausethereareothersourceofMDA.DPPP,Diphenyl-1-pyrenylphosphine,wasdevelopedbyDr.Meguro,et al. asafluorescentprobefordetectinglipidperoxide.ItselectivelyreactswithhydroperoxidestogenerateDPPPoxidethatemitsfluorescenceat380nm(ex:352nm),andmakesitpossible toquantify0.1 to7nmolofhydroperoxide. Inaddition, therangeof1 to2pmolof lipidperoxidecanbeselectivelydetectedwith thecombinationofHPLCseparationandthepost-columnreactionwithDPPP.
DPPPDiphenyl-1-pyrenylphosphineC28H19P=386.42CASNo.[110954-36-4]Unit:10mg
1. General Protocol► HPLCdetectionofhydroperoxidesinplasmasample1. Prepare10mg/mlBHT(Butylhydroxytoluene)/CHCl3-Methanol(2:1)solution.2. Dissolveasamplein100µlofthesolutionabove.3. Add50µlofDPPPsolution(1mg/10mlinCHCl3:Methanol=1:1)tothesolution
preparedinstep2.4. Incubatethesolutionunderdarkconditionfor60minutesat60°C.5. CooldownthesolutionandmeasurethefluorescenceintensitybyHPLC.
► Determinationofhydroperoxidesoncellmembrane(in vivo)1. DisolveDPPPinDMSOandprepare5mMDPPP/DMSOsolution.2. Add thesolutionabove to cell suspension(1 x107 cells/ml) to the final
concentrationof50μMDPPP.3. Incubatethecellsat37°Cfor10minutes.4. WashthecellstwicewithHank'ssolution.5. Stimulate thecellsbyaddingH2O2orMethyl linoleatehydroperoxideand
measureflueorescenceintensity.
2. References1. K.Akasaka,et al.,AnAromaticPhosphineReagentfortheHPLC-fluorescenceDeterminationofHydroperoxides-Determi-
nationofPhosphatidylcholineHydroperoxidesinHumanPlasma.Anal Lett.1988;21:965-975.2. K.Akasaka,et al.,Normal-phaseHigh-performanceLiquidChromatograohywithaFluorimetricPostcolumnDetection
SystemforLipidHydroperoxides.J Chromatogr A.1993;628:31-35.3. Y.Okimoto,et al.,ANovelFluoresceintProbeDiphenyl-pyrenylphosphinetoFollowLipidPeroxidationinCellMembranes.
FEBS Lett.2000;474:137-140.
3. Specification► Appearance:slightlyyellowpowder► Purity:≥97.0%(HPLC)
4. Recent Publications
DetectionoflipidperoxidebyHPLC
DPPP Product Code: D350
Fig. 1 Reaction scheme of DPPP with hydroperoxide
Title ReferenceDysregulationofverylongchainacyl-CoAdehydrogenasecoupledwithlipidperoxidation Y.Kabuyama,et al., Am J Physiol Cell Physiol.2010;298:
C107-C113.
CellDeathCausedbySeleniumDeficiencyandProtectiveEffectofAntioxidants Y.Saito,et al., J. Biol. Chem.2003;278:39428-39434.
Inductionof1-cysperoxiredoxinexpressionbyoxidativestressinlungepithelialcells Han-SukKim,et al., Am J Physiol Lung Cell Mol Physiol.2003;285:L363-L369.
AnAntisenseOligonucleotide to1-cysPeroxiredoxinCausesLipidPeroxidationandApoptosisinLungEpithelialCells
JhangHoPak,et al., J. Biol. Chem.2002;277:49927 -49934.
1-Cysperoxiredoxinoverexpressionprotectscellsagainstphospholipidperoxidation-mediatedmembranedamage YefimManevich,et al., PNAS.2002;99:11599-11604.
1. A
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L=Lipid
Dojindo Oxidative Stress Products
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DetectionofproteinorDNAradicalbyWB,ELISA,andimaging
DMPO Product Code: D048
Immuno-spinTrappingmethodwasdevelopedfordetectingDNAandProteinradicalsinbiologicalanalysis.ROS(ReactiveOxygenSpecies)producesmodificationofthestructureandfunctionofbiomoleculesthatrelateonthecauseofvarietydiseases.Tounderstandthemechanismofoxidativereactions,itisveryimportanttoanalyzewhichmoleculesareinvolvedintheoxidationprocess.DMPOisthemostpopularspin-trappingreagentthattrapsradicalsinproteinandDNAsamples.TheDMPO-ProteinorDMPO-DNAnitroneadductsaredeterminedusingaELISA,WesternBlotting,MassSpectorometry,Imaging,andsoon.ThepurityofDojindo’sDMPOishigherthananothercommercializedDMPO.Sinceitdoesnotcontainimpuritiesthatmightcausehighbackground.Dojindo’sDMPOdoesnotrequireanypre-purificationsteps.
DMPO5,5-Dimethyl-1-pyrrolineN-oxideC6H11NO=113.16CASNo.[3317-61-1]Unit:1ml
Fig. 1 Principle of Immuno-spin Trapping Method
1. Protocol Example : Radical DNA DetectionReferred PublicationDetectionandimagingofthefreeradicalDNAincells--site-specificradicalformationinducedbyFentonchemistryanditsrepairincellularDNAasseenbyelectronspinresonance,immuno-spintrappingandconfocalmicroscopy.BhattacharjeeS,ChatterjeeS,JiangJ,SinhaBK,MasonRP.,Nucleic Acids Res. 2012,40,5477-86
► EvaluationofradicalDNAbyELISA1. TreateRAWcells(1-3x106cells)with100µMCuCl2,100µMH2O2,and100
mMDMPOandincubatefor12-15hoursat5%CO2incubator.2. ExtractDNAfromRAWcellsanddiluteDNAto5μg/mlinPBS.3. Add25μlofDNAsolutionand25μlofReacti-BindDNAcoatingsolutionineach
welloftheplateandincubatefor2–4hoursat37°C.4. Washthewellsoncewithwashingbuffer(PBScontaining0.05%non-fatdry
milkand0.1%Tween-20).5. Blockwithblockingbuffer(PBScontaining3%non-fatdrymilk)for2hoursat37°C.6. DetectDMPO-DNAradicaladductwithanti-DMPOantibodyandHRP-conjugated
secondaryantibody.7. Afterthreewashes,addtheImmobilonchemiluminescencesubstrateeachwell
andmeasuretheintensityofluminesscense.► Anotherapplicationinthispaper
CellImaging
2. Protocol Example : Radical Protein DetectionReferred PublicationSuperoxideinducesendothelialnitric-oxidesynthaseproteinthiylradicalformation,anovelmechanismregulatingeNOSfunctionandcoupling.ChenCA,LinCH,DruhanLJ,WangTY,ChenYR,ZweierJL.,J Biol Chem.2011,286,
► Evaluationofradicalproteinbycellimaging1. PrepareBovineaorticendothelialcells(104cells)in35-mmdishes.2. Add10μMMenadioneand50mMDMPOandincubatecells.3. WashthecellswithPBSandfixthemwith3.7%paraformaldehydefor10minutes.4. Permeabilizethecellswith0.25%TritonX-100inTBST(Trisbufferedsalinewith
Tween)for10minutesandblockthecellswith5%goatseruminTBST.5. VisualizeDMPO-proteinradicaladductwithanti-DMPOantibodyandfluorescein
labeledsecondaryantibody.6. Analyzeproteinradicalsbyfluorescentmicroscopy.► Otherapplicationsinthispaper
Immunoblotting,MassSpectrometry,Immunoprecipitation
3. Specification► Appearance:colorlessliquid► Purity:≥99.0%(GC)
Fig. 2 Purity comparison in HPLC spectra
(*:impurities)
Dojindo
supplierS.
supplierE.
Fig. 3 Radical Detection Scheme
Radical
DNAorProtein
anti-DMPOPrimaryAntibody
FluoresceinorEnzymeLabeledSecondaryAntibody
DNA,Protein
DNARadicalProteinRadicallifetime:μsec.tosec.
DMPO-DNARadicalAdductsDMPO-ProteinRadicalAdductslifetime:min.tohr.
DMPO-DNAnitroneAdductsDMPO-ProteinnitroneAdductslifetime:monthstoyear
Oxidants
DMPO
-e-,-H+
DetectionMethod
EPR
EPR-spinTrapping
Immuno-spinTrapping• ELISA• CellImaging• Immunoblotting• Massspectrometry
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch► ESRspectrum:topasstest► IRspectrum:authentic
Dojindo Oxidative Stress Products
16
DetectionofradicalbyEPR
DMPO Product Code: D048
Becauseofpotentialcancerrisksandtheirage-promotingeffects,freeradicalsinliv-ingbodieshavebecomeafrequentlystudiedsubject.DMPOisthemostfrequent-
lyusedspin-trapping reagent for thestudyof free radicals. It issuitable for trappingoxygenradicals,especiallysuperoxides,andforproducingadductswithcharacteristicEPR(ESR)patterns.However,mostcommerciallyavailableDMPOcontainsimpuritiesthatcausehighbackgrounds.Thus,DMPOrequiresfurtherpurificationwhenrunningexperimentsonEPR.ThequalityofDojindo’sDMPOiswellcontrolledandDojindo’sDMPOdoesnotrequireanypre-purificationprocess.Therearenoimpuritiestocauseabackgroundproblem.
DMPO5,5-Dimethyl-1-pyrrolineN-oxideC6H11NO=113.16CASNo.[3317-61-1]Unit:1ml
Fig. 1 ESR Spectera of DMPO Adducts
1. Protocol► Evaluationofsuperoxidescavengingactivities1. Add15µlofDMPOand50µlof5mMhypoxanthineto35µlof0.1MPhosphate
buffer(pH7.8).2. Add50µlofSODstandardorsamplestobetestedandvoltexfor1-2seconds.3. Add50µlof0.4U/mlxanthineoxidaseandvolteximmediately.4. TransferthesolutiontoESRsampletubeandmeasureESRspectraaftercertain
timeofperiod,e.g.1minute.5. Calculaterelativeintensity(DMPO-O2
-/Mn2+)fromthepeakheight.
► C-,N-,andS-centeredradicalsDetection1.Prepareasolutionof100mMphosphatebuffer(pH7.4)containing25µMDTPA.2.Makeupasolutionofthefollowingperoxidasesubstrates:(A)100mMsodiumformate
(HCOONa);(B)100mMpotassiumcyanide(KCN);(C)100mMsodiumazide(NaN3);(D)100mMsodiumsulfite(Na2SO3)in100mMphosphatebuffer,pH7.4.
3.Makeupasolutionofhorseradishperoxidasewithconcentrationof4.0mg/ml(~100µM)and1mMsolutionofhydrogenperoxide(H2O2).
4.MakeupasolutionofDMPOwithconcentrationof1M.5.Prepareyourreactionmixturetoatotalreactionvolumeof200µlandadd130µlof
buffertoanEppendorftube.6.Add20µlDMPOofyour1MDMPOsolution,20µlofoneofthesubstrates’stock
solutions,10µlof1mMH2O2,andinitiatethereactionwith20µlHRP.7.Vortexthetube,transferthesolutiontoaflatcell,andacquirethespectrum.8.Thefinalconcentrationsofthecomponentsare:100mMDMPO,10mMsubstrate
(formate,cyanide,azide,sulfite),50µMH2O2,and10µMHRP. *ThisprotocolwaskindlyprovidedbyBrukerCorporation.
2. SpecificationRefertopreviouspage(Page15)
3. Recent Publications
Fig. 2 Purity comparison in HPLC spectra
(*:impurities)
Dojindo
supplierS.
supplierE.
Dojindo
supplierS.
supplierE.
Fig. 3 Purity comparison of ESR spectra
(black:fentonreaction,blue:blank)
Title ReferenceNonenzymaticdisplacementofchlorineandformationoffreeradicalsuponthereactionofglutathionewithPCBquinones
YangSong,BrettA.,andGarryR.Buettner,et al., PNAS.2009;106:9725-9730.
ManganeseSuperoxideDismutaseModulatesHypoxia-InducibleFactor-1InductionviaSuperoxide SuwimolKaewpila,andLarryW.Oberley,et al., Cancer Res.2008;68:2781-2788.
Hyperglycemia-InducedReactiveOxygenSpeciesToxicity toEndothelialCells IsDependentonParacrineMediators
JuliaV.Busik,andMariaB.Grant,et al., Diabetes,2008;57:1952-1965.
OverexpressionofExtracellularSuperoxideDismutaseAttenuatesHeparanaseExpressionandInhibitsBreastCarcinomaCellGrowthandInvasion.
MelissaL.T.,andFrederickE.Domann,et al., Cancer Res.,2009;69:6355-6363
SmokingInducesBimodalDNADamageinMouseLung "ShunjiUenoandKyosukeTemma,Toxicol. Sci.2011;120:322-330."
CardiacMyocyte–SpecificExpressionofInducibleNitricOxideSynthaseProtectsAgainstIschemia/ReperfusionInjurybyPreventingMitochondrialPermeabilityTransition
MatthewB.andAruniBhatnagar,et al., Circulation.2008;118:1970-1978.
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DetectionofsuperoxideradicalbyEPR
BMPO Product Code: B568
Spintrappinganalysisisoneofthemostreliabletechniquesfordetectingandiden-tifying short-lived free radicals.TheEPR (ESR) spin trap reagent detects both
superoxideandhydroxyl radicals producedby systems in vitro and in vivo. BMPOwasdevelopedasaspintrappingreagentthatadductssuperoxideandshowsamuchlongerhalf-life(t1/2=24min)thanotherspintrapreagents.Itgivesusreproducibleandsteadyresults.BecauseBMPOishighlysolubleinwater,hydrophilicsampleisappli-cableforanalyzingthefreeradicals.
BMPO5-tert-Butoxycarbonyl-5-methyl-1-pyrrolineN-oxideC10H17NO3=199.25Unit:50mg
1. General Protocol► MeasuringhydroxyradicalfromFentonreaction1. Add15µlofBMPOsolution,75µlof1mMH2O2and75µlof100µMFeSO4to
50µlofddH2O.2. TransferthesolutiontoESRsampletubeandmeasureESRspectraaftercer-
taintimeofperiod,e.g.1minute.3. Calculaterelativeintensityfromthepeakheight.
► Measuringsuperoxideradicalfromxanthineoxidase(XO)reaction1. Dissolve1mgofBMPOwith1mlof50mMPhosphatebuffer(pH7.4)(solutionA).2. Prepare50mMPhosphatebuffer(pH7.4)containing1mMDTPAand0.4mM
Xanthine(solutionB).3. Prepare50mMPhosphatebuffer(pH7.4) containing0.1U/mlxanthine
oxidase(solutionC).4. Mix15µlofsolutionA,135µlofsolutionBand10µlofsolutionC.5. TransferthesolutiontoESRsampletubeandmeasureESRspectraaftercer-
taintimeofperiod,e.g.8minutes.6. Calculaterelativeintensityfromthepeakheight.
2. References1. H.Zhao,J.Joseph,H.Zhang,H.KarouiandB.Kalyanaraman,Free Radic Biol Med.2001;31:599-606.2. G.M.Rosen,P.Tsai,J.Weaver,S.Porasuphatana,L.J.Roman,A.A.Starkov,G.FiskumandS.Pou,J Biol Chem.
2002;277:40275-40280.
3. Specification► Appearance:whitecrystalorcrystallinepowder► Purity:≥99.0%(HPLC)
Fig. 1 ESR Spectra of hydroxy radical adduct(black:fentonreaction,blue:blank)
Fig. 2 ESR Spectra of superoxide radical adduct(black:XOreaction,blue:blank)
Fig. 3 Half-life of BMPO-superoxide radical adduct
1591317212529333739Times(minutes)
1700
1500
1300
1100
900
700
500
300
100
PeakHeight
t1/2=24min
1. Anti O
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etection4. R
adical Detection
6. NO
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xide D
etection7. A
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2. References1. E.F.Ullman,et al.,StudiesofStableFreeRadicals.X.NitronylNitroxideMonoradicalsandBiradicalsasPossibleSmall
MoleculeSpinLabels.J Am Chem Soc.1972;94:7049-7059.2. Y.Miura,et al.,PolymersContainingStableFreeRaficals,5.PreparationofaPolymerContainingImidazoline3-Oxide
1-OxylGroups.Macromol Chem Phys.1973;172:233-236.3. K.Inoue,et al.,MagneticPropertiesoftheCrystalsofp-(1-Oxyl-3-Oxide-4,4,5,5-Tetramethyl-2-Imidazolin-2-Yl)Benzoic
acidandItsAlkaliMetalSalts.Chem Phys Lett.1993;207:551-555.4. T.Akaike,et al.,AntagonisticActionofImidazolineoxylN-OxidesAgainstEndothelium-DerivedRelaxingFactor/NOThrough
aRadicalReaction.Biochemistry.1993;32:827-832.5. J.Joseph,et al.,TrappingofNitricOxidebyNitronylNitroxides:anElectronSpinResonanceInvestigation.Biochem
Biophys Res Commun.1993;192:926-934.6. M.Yoshida,et al.,TherapeuticEffectsofImidazolineoxylN-OxideAgainstEndotoxinShockThroughItsDirectNitricOxide-
scavengingActivity.Biochem Biophys Res Commun.1994;202:923-930.7. T.Az-Ma,et al.,ReactionBetweenImidazolineoxilN-Oxide(Carboxy-PTIO)andNitricOxideReleasedfromCulturedEndo-
thelialCells:QuantitativeMeasurementofNitricOxidebyESRSpectrometry.Life Sci.1994;54:PL185-PL190.8. H.Maeda,et al.,MultipleFunctionsofNitricOxideinPathophysiologyandMicrobiology:AnalysisbyaNewNitricOxide
Scavenger.J Leukoc Biol.1994;56:588-592.
3. Specification► Appearance:darkbluepowder► Purity:≥97.0%(TLC)
4. Recent Publications
Fig. 2 ESR Spectra of Carboxy-PTIO and Carboxy-PTI
Carboxy-PTIOisastable,water-solubleorganicradicalthatreactswithNOtoformNO2·Thisreactioncanbemonitoredbyelectronspinresonance(ESR).NOisan
unstablemoleculeandhasacomplexreactioncascadeforitsmetabolisminbiologicalsystems.Rapidly generatedNO-relatedmetabolites carryout variousphysiologicalactivities.Commonly usedNOscavengers suchashemoglobin trapNO; theyalsotrapNOS inhibitorssuchasargininederivatives.TheseNOscavengersalsoquenchallotherNO-relatedmetabolitesatthesametime.Incontrast,Carboxy-PTIOdoesnotdramaticallyaffectotherNO-relatedproductsystemsbecauseittransformsNOtoNO2,whichisametaboliteofNO.Thus,Carboxy-PTIOcanbeusedtoinvestigatetheeffectsofNOseparately from its downstreammetabolites.Dr.Akaikeandothers showedthatCarboxy-PTIOsuppresses relaxationof the rat aorta ring,which is inducedbyacetylcholine,twiceaseffectivelyasNG-nitroarginine.Dr.Yoshidaandothersreportedthat downstreammetabolites ofNO,generatedby treatmentwithCarboxy-PTIO,havean increasedantiviralactivitycompared toNOalone.TheNOmetabolitesplayimportantrolesinbiologicalsystems;therefore,theyshouldbeinvestigatedseparatelyfromNO.
Carboxy-PTIO2 - ( 4 -Ca rboxypheny l ) - 4 , 4 , 5 , 5 - t e t r ame thy -limidazoline-1-oxyl-3-oxide,sodiumsaltC14H16N2NaO4=299.28CASNo.[148819-93-6]Unit:10mg
NOScavenger,NOdetectionbyEPR
Carboxy-PTIO Product Code: C348
Fig. 1 Reaction of NO trapping
Carboxy-PTIO
Carboxy-PTI
Title Reference
EffectofHypoxiaonSusceptibilityofRGC-5CellstoNitricOxideTakakiSato andTsunehiko Ikeda,et al., Invest. Ophthalmol. Vis. Sci.2010;51:2575-2586.
UpregulationofNitricOxideProductioninVascularEndothelialCellsbyAll-transRetinoicAcidThroughthePhosphoinositide3-Kinase/AktPathway
Akira Uruno and Sadayoshi Ito, et al., Circulation.2005;112:727-736.
TheNitricOxide–cGMPPathwayControls theDirectionalPolarityofGrowthConeGuidanceviaModulatingCytosolicCa2+Signals
TakuroTojimaandHiroyukiKamiguchi,et al., J. Neurosci.2009;29:7886-7897.
NitricOxideTransientlyConvertsSynapticInhibitiontoExcitationinRetinalAmacrineCells BrianHoffpauirandEvannaGleason,et al., J Neurophysiol.2006;95:2866-2877.
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TheGriessassay isasimpleandpopularmethod fordetectingNOconcentration.2,3-Diaminonaphthalene (DAN) is a highly sensitivealternative to theGriess
assay.TheDANmethod is 50-100 timesmore sensitive than theGriessassay:While thedetection limit of theGriessassay is 1mM, the limit of theDANmethodis 10-50nM.DAN reactswithNO2
- in acidic conditions to produce fluorescentnaphthalenetriazole.Thewavelengthoftheemissionmaximumofnaphthalenetriazoleis410nm.However,detectionat450nmisrecommendedtoavoidfluorescentblanksand increase sensitivity.The fluorescent backgroundofDAN is low formaximumsensitivity.Theoptimal reactionconditionsofDANwithNO2
-havebeendetermined.The reaction shouldproceedat pH2at room temperature for 5minutes, and theresultingfluorescenceofnaphthalenetriazoleshouldbedeterminedatapHof10ormore.DANisaphotosensitivereagentandsometimesbecomesdarkbrowncoloredcrystals.Since this brownproduct cannot beutilized for the fluorescent detection,recrystallizationisnecessary.
2,3-Diaminonaphthalene(for NO detection)2,3-DiaminonaphthaleneC10H10N2=158.20CASNo.[771-97-1]Unit:10mg
DetectionofNO2-byfluorometricanalysis
2,3-Diaminonaphthalene (for NO detection) Product Code: D418
1. General Protocol1. Dissolve50µgDANin1ml0.62MHCltoprepare0.31mMDANsolution.a)2. Mix10µlDANsolutionwith100µlNaNO2solution(0-10mM)orsamplesolu-
tion.3. Incubatethemixtureatroomtemperaturefor10-15minutes.4. Add5µl2.8MNaOHsolutiontothereactionsolution.b)5. Dilute100µlofthissolutionwith4mlwater,followedbyfluorescentmeasure-
mentwithexcitationwavelengthat365nmandemissionwavelengthat450nm.
6. PrepareacalibrationcurveusingthisdatawheretheX-axisisNaNO2concen-trationandtheY-axisisfluorescenceintensity.ThenusethiscalibrationcurvetodeterminetheNO2concentrationofthesamplesolution.a) Acidicconditionsarerequiredforarapidreaction.b) Basic conditions (pH10or higher) are required for a high fluorescence
signal.
2. References1. W.R.Tracey,et al.,Comparisonofspectrophotometricandbiologicalassaysfornitricoxide(NO)andendothelium-
derivedrelaxing factor (EDRF):nonspecificityof thediazotizationreaction forNOand failure todetectEDRF.J Pharmacol Exp Ther.1990;252:922-928.
2. J.S.Pollock,et al.,Purificationandcharacterizationofparticulateendothelium-derivedrelaxingfactorsynthasefromculturedandnativebovineaorticendothelialcells.PNAS.1991;88:10480-10484.
3. S.Archer,Measurementofnitricoxideinbiologicalmodels.FASEB J.1993;7:349-360.4. J.H.Wiersma,2,3-DiaminonaphthaleneasaSpectrophotometricandFluorometricReagentfortheDeterminationof
NitriteIon.Anal Lett.1970;3:123-132.5. C.R.Sawicki,FluorimetricDeterminationofNitrate.Anal Lett.1971;4:761-775.6. P.Damiani,et al.,Fluorometricdeterminationofnitrite.Talanta.1986;8:649-652.7. T.P.Misko,et al.,A fluorometricassay for themeasurementofnitrite inbiological samples.Anal Biochem.
1993;214:11-16.8. G.L.Wheeler,etal.,Rapiddeterminationoftraceamountsofselenium(IV),nitrite,andnitratebyhigh-pressureliquid
chromatographyusing2,3-diaminonaphthalene.Microchem J.1974;19:390-405.
3. Specification► Appearance:whiteorpaleyellowish-brownpowder► MeltingPoint:185°Cto200°C
4. Recent PublicationsTitle Reference
RoleoftheSphingosineRheostatintheRegulationofCnidarian-DinoflagellateSymbioses OlivierDetournay,Biol. Bull.2011;221:261-269.
TAK-242(Resatorvid),aSmall-MoleculeInhibitorofToll-LikeReceptor(TLR)4Signaling,BindsSelectivelytoTLR4andInterfereswithInteractionsbetweenTLR4andItsAdaptorMolecules
N.Matsunaga,et al., Mol. Pharmacol.2011;79:34-41.
ANovelCyclohexeneDerivative,Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), Selectively InhibitsToll-LikeReceptor4-MediatedCytokineProductionthroughSuppressionofIntracellularSignaling
MIi,et al., Mol. Pharmacol.2006;69:1288-1295.
1. Anti O
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etection2. D
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Diethyldithiocarbamate (DETC) is a good spin-trapping reagent for nitric oxide invivo.However,DETChasnotbeenwidelyutilized forNOdetection inbiological
samplesdue to its poorwater solubility.DTCS,ananalogofDETC, formsawater-solubleiron(II)complex(Fe-DTCS).TheFe-DTCScomplexthenformsacomplexwithNO(NO-Fe-DTCS).Dr.Yoshimurasuccessfullyobtainedtwo-dimensionalESRimagesofNO,inducedbylipopolysaccharideinmouseperitoneum.DTCSsodiumsalt(DTCSNa)wasusedforthisexperimentbecauseitislesstoxicthanammoniumsalt(sodiumsaltLD50:1942mg/kg;ammoniumsaltLD50:765mg/kg).SincetheFe-DTCScomplexismore stable than theother dithiocarbamate complexes in theair or in aqueoussolutions,itcouldbeausefulspintrappingreagentforbiochemicalresearch.TheFe-DTCScomplexshouldbeusedimmediatelyafterpreparation.AnexcessiveamountofDTCSNa(usually5equivalentsDTCSNatoFeSO4)isrequiredtomakeamorestablesolution.Dithiocarbamatestendtodecomposeunderphysiologicalconditionstoformtoxiccarbondisulfide.
DTCS NaN-(Dithiocarboxy)sarcosine,disodiumsalt,dihydrateC4H5NNa2O2S2•2H2O=245.23CASNo.[13442-87-0Unit:100mg,500mg
2. References1. T.Yoshimura,et al.,InvivoEPRDetectionandImagingofEndogenousNitricOxideinLipopolysaccharide-treatedMice.
Nat Biotechnol.1996;14:992-994.2. B.Kalyanaraman,DetectionofNitricOxidebyElectronSpinResonanceinChemical,Photochemical,Cellular,Physiologi-
cal,andPathophysiologicalSystems.Methods Enzymol.1996;268:168-187.3. H.Yokoyama,et al., InvivoESR-CTImagingoftheLiver inMiceReceivingSubcutenousInjectionofNitricOxide-Bound
IronComplex.Magn Reson Imaging.1997;15:249-253.
3. Specification► Appearance:whiteorpaleyellowpowder
DetectionofNObyEPR
DTCS Na Product Code: D465
1. General Protocol► PreparationofFe(II)-DTCSComplex1. Dissolve278mgFeSO4,7H2O(ferroussulfateheptahydrate)with20mlwa-
tera)toprepare50mMFeSO4solution.b)
2. Dissolve123mgDTCSNawith10mlwatera)toprepare50mMDTCSsolu-tion.
3. Mix1mlDTCSNasolutionwith8.8mlbuffersolutiona)(pH7orhigher).Add200µlFeSO4solutionjustpriortouse.
c)
a) Purgeanydissolvedoxygen in thewater or thebuffer bynitrogengasbubblingforatleast30minutespriortodissolvingFeSO4.
b) TheFeSO4solutioncanbestoredat-20°Cforatleast2months.c) Fe(II)-DTCScomplex is colorless. If the solution isbrown,Fe(III)-DTCS
mayhave formedbydissolvedoxygen in the solution.However, thebrownsolutioncanstillbeusedforNOtrapping.
► PreparationofNO-Fe(II)-DTCSComplex1. Underargongasflow,add200µlofFeSO4solutionto9.4mlbuffersolution(pH
7orhigher).ThenintroduceNOintothesolutionbybubblingNOgasthroughaglasscapillaryfor15minutes.
2. Add400mlofDTCSNasolutiontotheFeSO4solution,andcontinuetointro-duceNObybubblingforanother5minutes.
3. RemoveexcessNOwithargongasbubblingfor5minutes,andstoreat-20°C.TheNO-Fe(II)-DTCSsolutioncanbestoredat-20°Cforatleast2monthsinoxygen-freeconditions.
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DetectionofNObyEPR
MGD Product Code: M323
MGD isahighlywater-solubledithiocarbamate-typechelator that generateswi th complexes many t rans i t iona l method such as Fe and Cu. The
diethyldithiocarbamate-Fe2+ complexhasbeenused forNOdetectionbyelectronspin resonance (ESR).However, thepoorsolubilityof thiscarbamate inanaqueoussolutionlimitsitsapplication.Dr.Laiandothersimprovedthetechniqueusingawater-solubledithiocarbamate-Fe2+complex,MGD-Fe2+.TheysuccessfullydetectedinvivoNOof a nitroprusside-injectedmouseandNOgeneratedbyanLPS injectionusinginvivoESR.TheMGD-Fe2+complex iscapableofNOdetectionunderphysiologicalconditions,anddissolvedoxygeninthesolutiondoesnotinterferewithNOdetection.
MGDN-(Dithiocarbamoyl)-N-methyl-D-gluca-mine,sodiumsaltC8H16NNa2O5S2=293.34CASNo.[94161-07-6(freeacid)]Unit:500mg
1. General Protocol► PreparationofFe(II)-MGDComplex1. Dissolve278mgFeSO4,7H2O(ferroussulfateheptahydrate)with20mlwa-
tera)toprepare50mMFeSO4solution.b)
2. Dissolve147mgMGDwith10mlwatera)toprepare50mMMGDsolution.3. Mix1mlMGDsolutionwith8.8mlbuffersolutiona)(pH7orhigher)andthen
add200µlFeSO4solutionpriortouse.c)
a) Purgeanydissolvedoxygen in thewaterorbufferbynitrogengasbub-blingforatleast30minutespriortodissolvingFeSO4.
b) TheFeSO4solutioncanbestoredat-20°Cforatleast2months.c) Fe(II)-DTCSiscolorless.Ifthesolutionisbrown,Fe(III)-DTCSmayhave
formedbecauseofdissolvedoxygeninthesolution.However,thebrownsolutioncanstillbeusedforNOtrapping.
► PreparationofNO-Fe(II)-MGDComplex1. Underargongasflow,add200µlFeSO4solutionto9.4mlbuffersolution(pH
7orhigher).ThenintroduceNOintothesolutionbybubblingNOgasthroughaglasscapillaryfor15minutes.
2. Add400µlMGDsolutiontotheFeSO4solutionandcontinuetointroduceNObybubblingforanother5minutes.
3. RemoveexcessNOwithargongasbubblingfor5minutesandstoreat-20°C.TheNO-Fe(II)-MGDsolutioncanbestoredat -20°Cforat least2months inoxygen-free conditions.RemoveexcessNOwith argongasbubbling for 5minutes, andstoreat -20°C.TheNO-Fe(II)-DTCSsolution canbestoredat-20°Cforatleast2monthsinoxygen-freeconditions.
2. Specification► Appearance:whitecrystallinepowder► Purity:≥98.0%(HPLC)
1. Anti O
xidant D
etection2. D
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Fig. 1 Time course of releasing NO from NOR compounds
NORsare idealNOdonorswith completely different chemical structures from theotherNOdonors.AlthoughNORsdonot haveanyONO2orONOmoiety, they
spontaneouslyreleaseNOatasteadyrate.EventhoughtheNOreleasemechanismofNORhasnot been completely determined, it is confirmed that thebyproductsdonot possessany significant bioactivities.NOR3, isolated fromStreptomycesgenseosporeus,isreportedtohavestrongvasodilatoryeffectsonratandrabbitaortasanddog coronaryarteries. Its activity (ED50=1nM) is 300 times that of isosorbidedinitrate(ISDN).NOR3alsoincreasestheplasmacyclicGMPlevels,whereasISDNdoesnot.NOR is a potent inhibitor of platelet aggregationand thrombus formation.NOR3 (IC50=0-7mM)effectively inhibits 100%ofADP-initiatedhumanplateletAggregation,whereasISDNinhibitsonly32%ofthetotalaggregation,evenat100mMconcentrations.NOR3hasalsobeenreportedtohaveantianginalandcardioprotectiveeffects in the ischemia/reperfusion system. In the ratmethacholin-induced coronaryvasospasmmodel,NOR3 suppressed theelevationof theST segment dose-dependentlyandsignificantlyat1mgperkg.Ontheotherhand,ISDNsuppresseditsignificantlyat3.2mgperkg.ThedifferenceintheNOreleaserateofNORreagentswasreflectedevenontheinvivohypotensiveeffects.NORmayalsobeusedorallyina0.5%methylcellulosesuspension.NORisrelativelystable inDMSOsolution.NOR1,whichhas the shortest half-life, is a promising reagent formakingNOstandardsolutionsforcalibration.Forthepreparationofthestandardsolution,apreciselydilutedNOR1/DMSOsolutionisaddedtothebuffersolutions.
NOR 1 (Product code: N388)(±)-(E)-4-Methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamideC8H13N3O5=231.21CASNo.[163032-70-0]Unit:10mg
NOR 3 (Product code: N390)(±)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamideC8H13N3O4=215.21CASNo.[163180-49-2]Unit:10mg
NOR 4 (Product code: N391)(±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridinecarboxamideC14H18N4O4=306.32CASNo.[162626-99-5]Unit:10mg
NOR 5 (Product code: N448)(±)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-6-methyl-5-nitro-3-heptenyl]-3-pyridine-carboxamideC16H22N4O4=334.37Unit:10mg
1. References1. S.Shibata,et al.,CharacteristicsoftheVasorelaxingActionof(3E)-4-Ethyl-2-hydroxyimino-5-nitro-3-hexamideFK409,a
NewVasodilatorIsolatedfromMicrobialSources,inIsolatedRabbitArteries.J Cardiovasc Pharmacol.1991;17:508-518.2. Y.Kita,et al.,AntianginalEffectsofFK409,aNewSpontaneousNOReleaser.Br J Pharmacol.1994;113:1137-1140.3. Y.Kita,et al.,AntiplateletActivitiesofFK409,aNewSpontaneousNOReleaser.Br J Pharmacol.1994;113:385-388.4. Y.Kita,et al.,SpontaneousNitricOxideReleaseAccountsforthePotentPharmacologicalActionsofFK409.Eur J Pharma-
col.1994;257:123-130.5. M.Hino,et al.,FK409,aNovelVasodilatorIsolatedfromtheAcid-treatedFermentationBrothofStreptomyces Griseo-
sporeusI.Taxonomy,Fermentation,Isolation,andPhysico-chemicalandBiologicalCharacteristics.J Antibiot.1989;42:1578-1583.
6. J.Decout,et al.,DecompositionofFK409,aNewVasodilator:IdentificationofNitricOxideasMetaborite.Bioorg Med Chem Lett.1995;5:973-978.
7. S.Fukuyama,et al.,ANewNitricOxide(NO)Releaser:SpontaneousNOReleasefromFK409.Free Radic Res.1995;23:443-452.
8. Y.Kita,et al.,FR144420,aNovel,Slow,NitricOxide-releasingAgent.Eur J Pharmacol.1995;275:125-130.9. M.Kato,et al.,NewReagentsforControlledReleaseofNitricOxide.Structure-stabilityRelationships.Bioorg Med Chem
Lett.1996;6:33-38.10. Y.Kita,et al.,FK409,aNovelSpontaneousNOReleaser:ComparativePharmacologicalStudieswithISDN.Cardiovasc
Drug Rev.1996;14:148-165.11. Y.Hirasawa,et al.,AntianginalEffectsofFR144420,aNovelNitricOxide-releasingAgent.Eur J Pharmacol.1996;303:55-
59.12. M.Sato,et al.,NitricOxideRaisesCytosolicConcentrationsofCa2+inCulturedNodoseGanglionNeuronsfromRabbits.
Neurosci Lett.1996;206:69-72.13. Y.Kita,et al.,OralBiologicalActivitiesofSpontaneousNitricOxideReleaserareAccountedforbytheirNitricOxide-
releaseingRatesandOralAbsorptionManners.J Pharmacol Exp Ther.1996;276:421-425.
2. Specification► Appearance:Whiteorslightlyyellowpowder(NOR1,NOR4,NOR5),Whitecrystallinepowder(NOR3)
► Purity:≥98.0%(HPLC)
NOdonor(Stableinacidiccondition)
NOR Compounds
NODonorsNOR1 NOR3 NOR4 NOR51.8min 30min 60min 20hrs
Table 1 Half-life of NOR donors(pH 7.4)
▲ ▲
0 15 30Time(min)
2.0
1.5
1.0
0.5
0
Conc.ofreleasedNO(mM)
▲
▲
▲▲
▲
▲
●
●
●●●
060120180240Time(min)
1.0
0.8
0.6
0.4
0.2
0
Conc.ofreleasedNO(mM)
▲
▲
▲
▲
▲
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NOCsare stableNO-amine complexes that spontaneously releaseNO,withoutcofactors, under physiological conditions.The rate ofNO releasedependson
the chemical structureofNOC.Themechanismof spontaneousNOgenerationbyNOCs is very simple compared to other classicalNOdonors, suchasnitroglycerinandnitropurusside, and theby-products donot interferewith cell activities.A singleNOCmolecule releases twoNOmolecules (as indicated in the reaction scheme);thereleaserateof thesecondNOmolecule isveryslow.NOCscanbeused toaddcontrolled amounts of pureNO toexperimental systemsat controlled rateswithminimalsideeffects.TheamountofNOreleasedcanbeeasilymanipulatedbyalteringtheconcentrationandselectionofNOCreagents.Dojindooffers fourdifferentNOCs(NOC5, 7, 12, and18)with different halflifes.Stock solutionsofNOCprepared inalkaline solutions, suchasaqueousNaOH,are relatively stable.However,theNOCstocksolutionshouldbeusedwithinonedaybecauseitdegradesabout5%perday,evenat-20ºC.ThereleaseofNObeginsimmediatelyafteraddingthestocksolutiontoasamplesolution.
NOC 5 (Product code: N380)1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazeneC6H16N4O2=176.22CASNo.[146724-82-5]Unit:10mg,50mg
NOC 7 (Product code: N377)1-Hydroxy-2-oxo-3-(N-methyl-3-amino-propyl)-3-methyl-1-triazeneC5H14N4O2=162.19CASNo.[146724-84-7]Unit:10mg,50mg
NOC 12 (Product code: N378)1-Hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazeneC6H16N4O2=176.22CASNo.[146724-89-2]Unit:10mg,50mg
NOC 18 (Product code: N379)1-Hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-tria-zeneC4H13N5O2=163.18CASNo.[146724-94-9]Unit:10mg,50mg
NOdonor(Stableinalkalinecondition)
NOC Compounds
NODonorsNOC5 NOC7 NOC12 NOC18
pH
7.0 12min 2.2min 40min 13hrs7.2 20min 3.8min 1.2hrs 18hrs7.4 25min 5min 100min 21hrs7.6 42min 8.2min 3hrs 34hrs7.8 66min 12.4min 4.6hrs 45hrs
Table 1 Half-life of NOC donors
1. General Protocol1. Prepare10mMNOCstocksolutionusing0.1MNaOH.SincetheNOCstock
solutionisnotstable,keepitonanicebathanduseitinoneday.2. AddanappropriatevolumeoftheNOCstocksolutiontothesamplesolution
inwhichNOistobereleased.TomaintainthepHofthesamplesolution,thevolumeoftheNOCstocksolutionshouldnotexceed1/50ofthesamplevolume.Thesamplesolutionshouldhavesufficientbufferingaction.NOwillbereleasedimmediatelyaftertheadditionoftheNOCstocksolution.
2. References1. K.Hayashi,et al.,ActionofNitricOxideasaAntioxidantAgainstOxidationofSoybeanPhosphatidylcholineLiposomal
Membrane.FEBS Lett.1995;370:37-40.(Noc12)2. S.Shibuta,et al.,IntracerebroventricularAdministrationofaNitricOxide-releasingCompound,NOC-18,ProducesThermal
HyperalgesiainRats.Neurosci Lett.1995;187:103-106.(NOC18)3. S.Shibuta,et al.,Anewnitricoxidedonor,NOC-18,exhibitsanociceptiveeffectintheratformalinmodel.J Neurol Sci.
1996;141:1-5.(NOC18)4. N.Yamanaka,et al.,NitricOxideReleasedfromZwitterionicPolyamine/NOAdductsInhibitsCu2+-inducedLowDensity
LipoproteinOxidation.FEBS Lett.1996;398:53-56.(NOC5,NOC7)5. D.Berendji,et al.,NitricOxideMediatesIntracytoplasmicandIntranuclearZincRelease.FEBS Lett.1997;405:37-41.6. T.Ohnishi,et al.,TheEffectofCu2+onRatPulmonaryArterialRings.Eur J Pharmacol.1997;319:49-55.(Noc7)7. Y.Adachi,et al.,RenalEffectofaNitricOxideDonor,NOC7,inAnethetizedRabbits.Eur J Pharmacol.1997;324:223-226.
(Noc7)8. Y.Minamiyama,et al.,Effect ofThiolStatusonNitricOxideMetabolism in theCirculation.Arch Biochem Biophys.
1997;341:186-192.(NOC7)
3. Specification► Appearance:Whitepowder► Purity:≥90.0%(HPLC)
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch
Dojindo Oxidative Stress Products
24
Fig. 1 Time course of NOC degradation(100 µM NOCs in 100 mM PBS, pH7.0, 37°C)
Fig. 2 Half life of NOC compounds in 100mM PBS(37°C) under the various pH conditions
Fig. 3 pH dependency of releasing NO from NOC 5(0.1 mM NOC 5, in 100 mM PBS at 37°C)
050100150200Time(min)
NO(μ
mol/l)
200
100
0
pH7.0(NO)pH7.2(NO)pH7.4(NO)pH7.6(NO)
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NOC5 NOC7
NOC12 NOC18
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ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ77.27.47.67.8
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77.27.47.67.8ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ
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NOC(μ
mol/l)
NOC(μ
mol/l)
time(min) time(hrs)020406080100120 05101520253035404550
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NOC5(UV250nm)NOC7(UV250nm)
1. A
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oxid
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adic
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O D
onor
5. N
itric
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Dojindo Oxidative Stress Products
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NNitrosothiol compounds releaseNOandbecomedisulfidesunder specificphysiologicalconditions.WhilemostoftheS-nitrosothiolcompoundsareunstable,
S-Nitrosoglutathione is exceptionally stable.Furthermore,S-Nitrosoglutathione iswater-soluble.AlthoughS-nitrosothiol is a goodNOdonorwith nonitrate tolerance,thereisevidencethatS-nitrosothiolitselfhasNO-likeactivityduringguanylatecyclaseactivation.Another important reactionof nitrosothiol isNO transfer to other thiolcompounds.Since itdependson thepKaof thiols, this transfer reactionproceedsatphysiologicalpHlevels.Previous research found that the vasorelaxant propertiesof endothelium-derived re-laxation factor (EDRF)aremore similar toS-nitrosocysteine thanNO;however, thisdoesnotseemtobethecurrentmajorityview.Thoughnitrosothiolisoneofthemostimportant factors for the studyof theNOpathway, only a fewnitrosothiols, suchasSNAPandS-nitrosoglutathione(GSNO),arestableenoughforuseasNOdonors.Un-fortunately,SNAPisinsolubleinwater.Thus,GSNOandS-nitrosocysteine(SNC)aretheonlycommerciallyavailablewater-solublenitrosothiols.Nitrosothiolsreleasenitricoxide,andformdisulfidesasshownbelow. 2RSNO→RSSR+2NO
This reaction isacceleratedby lightandheat. IfGSNO is incubatedat37°Cwithoutlight,NOwill not be released spontaneously.Metal ions, suchasCu(II),Cu(I), andHg(II), alsoaccelerate the reaction.Thus,masking reagents suchasEDTApreventthe releasing reaction.Another important characteristic of nitrosothiols is their abilitytocarryoutnitrosation.ThisreactionisfasterthanthedecompositionofRSNOitself,andproceedsreadilyatphysiologicalpHlevels.ThereactionratedependsonthepKaofthethiol.Thevasorelaxantactivitiesofnitrosothiolsinrataorticringshavebeenre-portedasfollows: SNAP>GSNO=SNAC(S-Nitroso-N-acetylcysteine) >CoAsNO(S-Nitroso-coenzymeA)>CYCNO(S-Nitrosocysteine)
Theinhibitorypotenciesofnitrosothiolsfortheplateletaggregationhavebeenreportedasfollows: GSNO>NO>SNAP>SIN-1
DenitrosationofS-nitrosothiolisnotspontaneous,anditneedstobecatalyzedonthesurfaceof external vascularmembranes.S-nitroso-L-cysteine raises the intracellularcalciumlevelofaPC12cellbymodifyingthethiolgroupofacaffeine-sensitivemoietyof the calcium-induced calcium release (CICR) channel.GSNOhasbeen shown toreducethebloodpressureofanesthetizeddogs(0.2mg/kg)andmonkeys(10mg/kg)throughtheinhibitionoftheplateletaggregation.
S-Nitrosoglutathione N-(N-L-γ-Glutamyl-S-nitroso-L-cysteinyl)glycineC10H16N4O7S=336.32CASNo.[57564-91-7]Unit:25mg,100mg
1. References1. A.Gibson,et al.,AnInvestigationofSomeS-Nitrosothiols,andofHydroxy-arginine,ontheMouseAnococcygeus.Br J
Pharmacol.1992;107:715-721.2. M.W.Radomski,et al.,S-Nitroso-glutathioneInhibitsPlateletActivationinVitroandinVivo.Br J Pharmacol.1992;107:745-
749.3. R.M.Clancy,et al.,NovelSynthesisofS-NitrosoglutathioneandDegradationbyHumanNeutrophils.Anal Biochem.
1992;204:365-371.4. J.W.Park,et al.,TransnitrosationasaPredominantMechanismintheHypotensiveEffectofS-Nitrosoglutathione.Bio-
chem Mol Biol Int.1993;30:885-891.5. D.Barrachina,et al.,NitricOxideDonorsPreferenciallyInhibitNeuronallyMediatedRatGastricAcidSecretion.Eur J
Pharmacol.1994;262:181-183.6. E.A.Konorev,et al.,S-NitrosoglutathioneImprovesFunctionalRecoveryintheIsolatedRatHeartAfterCardioplegicIsch-
emicArrest-evidenceforaCardioprotectiveEffectofNitricOxide.J Pharmacol Exp Ther.1995;274:200-2006.7. S.C.Askew,et al.,CatalysisbyCu2+ofNitricOxideReleasefromS-Nitrosothiols(RSNO).J Chem Soc Perkin Trans 2.
1995;741-745.8. D.J.Banett,et al.,NO-groupTransfer(Transnitrosation)betweenS-NitrosothiolsandThiols.Part2.J Chem Soc Perkin
Trans 2.1995;1279-1282.9. J.G.DeMan,et al.,EffectofCu2+onRelaxationstotheNitrergicNeurotransmitter,NOandS-NitrosothiolsintheRat
GastricFundus.Br J Pharmacol.1996;119:990-996.10. A.P.Dicks,et al.,GenerationofNitricOxidefromS-NitrosothiolsUsingProtein-boundCu2+Sources.Chem Biol.
1996;3:655-659.11. J.A.Cook,et al.,ConvenientColorimetricandFluorometricAssaysforS-Nitrosothiols.Anal Biochem.1996;238:150-158.12. S.X.Liu,et al.,NitricOxideDonors:EffectsofS-Nitrosoglutathioneand4-Phenyl-3-furoxancarbonitrileonOcularBlood
FlowandRetinalFunctionRecovery.J Ocul Pharmacol Ther.1997;13:105-114.13. C.Alpert,et al.,DetectionofS-NitrosothiolsandOtherNitricOxideDerivativesbyPhotolysis-chemiluminescenceSpec-
trometry.Anal Biochem.1997;245:1-7.14. T.Akaike,et al.,NanomolarQuantificationandIdentificationofVariousNitrosothiolsbyHighPerformanceLiquidChroma-
tographyCoupledwithFlowReactorsofMetalsandGriessReagent.J Biochem.1997;122:459-466.
2. Specification► Appearance:Pinkpowder► Purity:≥90.0%(HPLC)
NOdonorfromnitrosothiolcompound
S-Nitrosoglutathione Product Code: N415
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch
Dojindo Oxidative Stress Products
26
SIN-13-(4-Morpholinyl)sydnonimine,hydrochlorideC6H11ClN4O2=206.63CASNo.[16142-27-1]Unit:25mg
1. References1. M.Feelisch,et al.,OntheMechanismofNOReleasefromSydnonimines.J Cardiovasc Pharmacol.1989;14:S13-S22.2. M.Feelisch,TheBiochemicalPathwaysofNitricOxideFormationfromNitrovasodilators:AppropriateChoiceofExogenous
NODonorsandAspectsofPreparationandHandlingofAqueousNOSolutions.J Cardiovasc Pharmacol.1991;17:S25-S33.
3. N.Hogg,et al.,ProductionofHydroxylRadicalsfromtheSimultaneousGenerationofSuperoxideandNitricOxide.Bio-chem J.1992;281:419-424.
4. M.E.Murphy,et al.,NitricOxideHyperpolarizesRabbitMesentericArteriesviaATP-sensitivePotassiumChannels.J Physiol.1995;486:47-58.
5. H.Kankaanranta,et al.,3-Morpholino-sydnonimine-inducedSuppressionofHumanNeutrophilDegranulationinNotMedi-atedbyCyclicGMP,NitricOxideorPeroxynitrite:InhibitionoftheIncreaseinIntracellularFreeCalciumConcentrationbyN-Morpholinoiminoacetoni.Mol Pharmacol.1997;51:882-888.
6. S.Yamamoto,et al.,SubarachnoidHemorrhageImpairsCerebralBloodFlowResponsetoNitricOxidebutNottoCyclicGMPinLargeCerebralArteries.Brain Res.1997;757:1-9.
7. S.Pfeiffer,et al.,Interferenceofcarboxy-PTIOwithNitric-oxideandPeroxynitrite-mediatedReactions.Free Radic Biol Med.1997;22:787-794.
8. M.B.Herrero,et al.,TyrosineNitrationinHumanSpermatozoa:APhysiologicalFunctionofPeroxynitrite,theReactionProductofNitricOxideandSuperoxide.Mol Hum Reprod.2001;7:913-921.
9. P.D. Lu,et al.,CytoprotectionbyPre-emptiveConditionalPhosphorylationofTranslation InitiationFactor 2.EMBO J.2004;23:169-179.
2. Specification► Appearance:Whiteneedlesorslightlyyellowish-whitecrystallinepowder► Purity:passtest(TLC)► MeltingPoint:180°Cto190°C
SIN-1, ametabolite of the vasodilatormolsidomine, is utilized to separatelyestimate theeffectivenessofNOandperoxynitritewith otherNOdonors.SIN-
1 spontaneously decomposes in thepresenceofmolecular oxygen to generateNOandsuperoxide.Bothproductsbindveryrapidlytoformperoxynitrite(rateconstantk:3.7x10-7M-1s-1).Therefore,SIN-1isausefulcompoundthatgeneratesperoxynitriteinanefficientmanner.Peroxynitriteisaverystrongoxidantthatgenerateshydroxylandnitrosyldioxylradicalsunderphysiologicalconditions.Peroxynitritealsodecomposestogeneratenitrate ionquickly inacidicconditionsandslowly inbasicconditions.ThosespecieshaveadifferentbioactivityfromNO.
ONOO-(peroxynitrite)donor
SIN-1 Product Code: S264
1. A
nti O
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ectio
n2.
DN
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amag
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ipid
Per
oxid
e D
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4. R
adic
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6. N
O D
onor
5. N
itric
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ectio
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AG
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Dojindo Oxidative Stress Products
27
Advancedglycationend-products(AGEs)havebeenstudiedasoneofthecausesofdiabeticcomplications.SeveralcompoundshavebeenidentifiedasAGEs,including
pyralline,pentosidine, imidazolone,andpyropyridine.Glyoxalandmethylglyoxalarereactivedicarbonylcompoundsgeneratedbyglucoseself-oxidation thatareknowntobeAGEprecursors.Anotherdicarbonylcompound,3-Deoxyglucosone (3-DG),isalsoknowntobeoneof theAGEprecursors.3-DG isderived fromtheAmadorirearrangementproductsofproteinsandsugarsinearlystagesoftheMaillardreaction.3-DGisalsoderivedfromfructose,whichispresentinhighlevelsindiabeticpatients,byaselfcondensation reaction.Fructose-3-phosphatehasbeen found toenhancecross-linkingreactionsoflensproteinsinadiabeticratmodel.Therefore,3-DGderivedfromfructose-3-phosphatehasbeenstudiedasapossiblecauseofcataracts.Thereare twomethods fordetermining3-DGlevels:HPLCandmassspectrometry(MS).However,thereissomediscrepancybetweentheHPLCandMSmethodswhenmeasuring3-DGlevels invivo.HPLCanalysis isbasedonafluorescentcompound,2-(2,3,4-trihydroxybutyl)-benzo[g]quinoxaline,generatedbyacoupling reactionbetween3-DGand2,3-diaminonaphthalene.Analogsof2,3-diaminonaphthalene,suchas1,2-diamino-4,5-dimethoxy-benzeneand1,2-diamino-4,5-methylenedioxybenzene,canalsobeused.
Contents of the KitDAN[2,3-Diaminonaphthalene] 10mgx13-DG/DANadduct[2-(2,3,4-Trihydroxybutyl)benzo[g]quinoxaline] 1mgx1
3-DGcanbeutilizedforAGEproductionorasastandardfor3-DGleveldetectioninplasmaorserumsamples. 3-Deoxyglucosone
3-Deoxy-D-erythro-hexos-2-uloseC6H10O5=162.14CASNo.[4084-27-9]Unit:1mg
1. Specification► Appearance:whiteorwhitepaleyellowsolid► Purity:≥99.0%(HPLC)
2. Recent Publications
Detectionof3-DGbyHPLC(Fluorometric)
3-Deoxyglucosone Detection Reagents Product Code: D536
Fig. 1 Principal of 3-DG detection2. General Protocol► HPLCMethod:HumanSerum1. Add60%perchloricacidsolutionto1mlhumanserumandspinat3,000xg
for20minutesat4°C.2. Dilutethesupernatantwithbicarbonatebuffer,thenadd0.1mlof2,3-Diaminonaphthalene
/methanolsolutionand25µlof1ppm3,4-hexanedioneasaninternalstandard.3. Incubatethemixtureat4°Covernight.4. Extractthemixturewith4mlethylacetate,andadd4mlmethanoltotheextract.5. Analyzethemixturewithreverse-phaseHPLCat267nmexcitationand503
nmemissionforfluorescentdetectionorat268nmforUVdetection.DatacorrelateswellwithHbA1clevel.
*Normalserum3-DGlevel:12.8±5.2ng/ml *Serum3-DGlevelofdiabeticpatient:31.8±11.3ng/ml
3. References1. K.J.Knecht,et al.,Detectionof3-Deoxyfructoseand3-DeoxyglucosoneinHumanUrinendPlasma:EvidenceforInterme-
diateStagesoftheMaillardReactioninVivo.Arch Biochem Biophys.1992;294:130-137.2. T.Niwa,et al.,Presenceof3-Deoxyglucosone,aPotentProteinCrosslinkingIntermediateofMaillardReaction,inDiabetic
Serum.Biochem Biophys Res Commun.1993;196:837-843.3. H.Yamada,et al.,Increasein3-deoxyglucosonelevelsindiabeticratplasma.Specificinvivodeterminationofintermediate
inadvancedMaillardreaction.J Biol Chem.1994;269:20275-20280.
StandardsubstrateforAGEprecursor
3-Deoxyglucosone Product Code: D535
Title ReferenceGlutathionedepletionasamechanismof3,4-dideoxyglucosone-3-ene-inducedcytotoxicityinhumanperitonealmesothelialcells:roleinbiocompatibilityofperitonealdialysisfluids
T.Yamamoto,et al., Dial. Transplant.2009;24:1436-1442.
Anovelclassofadvancedglycation inhibitorsameliorates renalandcardiovasculardamageinexperimentalratmodels
Y. Izuhara,et al, Nephrol. Dial. Transplant.2008;23:497-509.
1. Anti O
xidant D
etection2. D
NA D
amage
Detection
3. Lipid Peroxide D
etection4. R
adical Detection
6. NO
Donor
5. Nitric O
xide D
etection7. A
GEs R
esearch