Special Micro Plate as Says 2009

Flash manuelle

Interchim and Berthold Technologies collaborationInterchim, a provider of consummables for life sciences, and Berthold Technologies, a leader in microplate instrumentation technology, have entered into a collaboration agreement to offer complete instrumentation and reagent solutions.

BERTHOLD TECHNOLOGIES provides with the Mithras and TriStar extremely versatile multimode readers for all comprehensive technologies used in today´s laboratory.

Additionally dedicated microplate readers for luminescence, fluorescence and absorbance can be offered for all common microplate formats. Petri dishes and Teraski plates can be measured with respective adapters.Powerful software allows kinetics, scanning, repeated mode, dual ratio measurements etc.

For higher troughput the instruments can be run with the Stacker LB 931. Robot access enables integration into robotic HTS systems.

Mithras LB 940

For instrument informations, please contact :

Berthold France SAS Parc Technologique des Bruyères

8, route des Bruyères 78770 Thoiry

France

Phone : (+33) 1 34 94 79 00 Fax : (+33) 1 34 94 79 01

E-mail : [email protected]

TriStar LB 941

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Reporter Gene Assays ........................................................................................................................................................................ 7 FireflyLuciferase 7 FireflyLuciferase1-StepAssayKit,2hreading,StableAssayKit,3-5hreading, FireflyLuciferase,recombinant,MammalianExpressionVectors:pCMVLuxA&pCMVLuxB,FireflyluciferasesiRNA constructs:siFLuc,pRNALuc/Neoformonitoringtranscriptionalactivity

RenillaLuciferase 10 RenillaLuciferaseAssayKit,RenillaMullereiLuciferase,recombinant,andpUC19plasmid(pRLuc)

FireflyandRenillaLuciferases 11 FireflyandRenillaLuciferasesAssayKit

GaussiaLuciferase 12 HumanizedGaussiaLuciferase,GaussiaLuciferaseAssaykit

Coelenterazines 13 Coelenterazine,native,CoelenterazineH(Benzyl-Coelenterazine),Coelenterazine400a

D-Luciferins 15 Substratesforglycosidasesreportersystems(β-galactosidase,β-glucuronidase,β-glucosidase) 16 β-GalactosidasefluorescentsubstratessamplerKit,Othercolorimetricsubstratesforglycosidases

Enzymes Detection ............................................................................................................................................................................ 17 Alkalinephosphataseassays 17 AlkalinePhosphataseAssayKit,Blue,Green,RedFluorescence,Luminometric

Acidphosphataseassays 18 MUPPlus

ProteaseAssayKits 19 LipaseassaysandLipidstudy 20 LipaseAssayKit,UnboundfreefattyacidsAssay

Collagenandelastinstudy 21 LysylOxidaseAssayKit,RedFluorescence

CellulaseDetection 21 CellulaseAssayKit,RedFluorescence

AcetylcholinesteraseDetection 22 aCella–AchEAcetylcholinesteraseAssay

CalciumAssays ................................................................................................................................................................................. 23 Singleλexc./em. Ca2+indicators 24 Fluo-3AM,Fluo-8NW,Fluo-8HAM,Fluo-8LAM;Rhod-4™,Rhod-4NWCalciumAssayKit

Ratiometriccalciumdyes 27 Fura-2AM,Fura-PE3AM,Indo-1AM,Indo-PE3AM

Viability&CytotoxicityAssays ........................................................................................................................................................ 28 Dualstainingtodetectliveanddeadcells 29 Live/DeadMammalianViabilityAssayKit,Live/DeadBacterialViability/Cytotoxicitykit,Ethidiummonoazide,bromide(EMA), Propidiummonoazide(PMA)

Livecellstaining 32 CalceinAMCellCounting&ViabilityAssayKit,CFDASE,UptiBlueCellViabilityAssayKit,WST-8CellProliferationandCytotoxicityAssayKit, ACella™-TOXBioluminescenceCytotoxicityAssay(GAPDH)

ATP,ADP,Phosphate&PyrophosphateAssays 36 ATPAssaykit,0.1to100pmol,ATPAssayKit,SteadyGlow,BrightGlow,UniversalFluorimetricKinaseAssayKit, ADPAssayKit,RedFluorescence,PhosphateAssayKit,PyrophosphateAssayKit,

NAD/NADH,NADP/NADPHAssayKits 41 NADHandNAD/NADHAssayKits

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Apoptosis Assays ............................................................................................................................................................................. 43 Caspasesstudy 43 CaspasesFluorometricHTSAssayKits,FluorimetricandColorimetricAssayKits,Caspase-6andGranzymeBbasedapoptosis/toxicityAssays

DNADamage&Condensationstudy 46 UniversalChemiluminescentPARPAssayKit,HTChemiluminescentPARP/ApoptosisAssay,HTChemiluminescentPARGAssayKit,FlowTACS™ ApoptosisDetectionKits,Hoechst33342

Mitochondrialevents 49 JC-10MitochondriaMembranePotentialAssayKit,SafranineO,GlutathioneDetectionKit,Monochlorobimane,LiveCellGlutathioneTransferaseActivityKit

ReactiveOxygenSpecies(ROS) 51 H2DCFDA,Carboxy-H2DCFDA,H2DCFDA-SE,Coelenterazine(native),MCLA,Dihydroethidium(hydroethidine),dihydrorhodamine-1,2,3(DHR123), DihydrocalceinAM,trans-1-(2'-Methoxyvinyl)pyrene,Hydroxyphenylfluorescein(HPF)AssayKit,HypochloriteDetectionKit,APF(forhROSdetection), ADHPHydrogenperoxideAssayKit,MyeloperoxidaseDetectionKit,CatalaseDetectionKit,Cis-ParinaricAcid(CPA),SODAssayKitwithcolorimetric substrateWST-1,Malachitegreenisothiocyanate

Aldehydeoxidationstudy 58 MonoamineOxidaseA&BDetectionKit,SSAODetectionKit

NitricOxide(NO)assay 59 DAF-2diacetate,DAF-FM,2,3-Diaminonaphthalene,NBDMethylhydrazinefornitriteassay

DNA,ProteinsandglucidesBiochemistryAssays ........................................................................................................................ 60 FluorescentDNAAssay 60 EvaGreen-DNAquantificationinsolution

Proteinassays 61 ColorimetricProteinassays–BCAssay,CooAssay,FluorescentProteinassays–LavaPep

Biochemistrytestsforbiologicalsamples 61

ImmunologicalAssays ...................................................................................................................................................................... 62 ELISA–Luminescentdetection(ECL) 62 UptiLightELISAHRPChemiluminescentSubstrate,LuminometricAlkalinePhosphataseAssayKit

OtherColorimetric&FluorimetricEnzymaticSubstratesforMicroplatesassays 64 UptimaTMBELISAchromogenicsubstrate,Substratesforhorseradishperoxidase,AlkalinePhosphatase

FluorescentsecondaryreagentsforELISA(IIAbs,Avidin) 66 Streptavidinconjugates,SecondaryAntibodiesconjugates

ImmunoAssay-Buffers&SaturatingAgents 68 StandardBuffers,ReadytouseBuffers,SeaBlocksaturatingagent(nonmammalianserum),Others

AntibodyLabelingKits 69 Microspinbiotinylationlabelingkits-NH2and–SH,FluoProbes®ProteinLabelingKits

Accessory tools ................................................................................................................................................................................. 71 FPlyteMicroplates 71 Bottom-freewellsmicroplates-IMAplateTechnology(IntelligentMultiFunctionalAnalysis) 72 AntifadeKitforMicroplate 75

Legals-Trademarks ................................................................................................................................................................................................. 75

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Firefly Luciferase

Interchimprovideskitsandstand-alonereagentstostudytheexpressionofLuciferasesfromdifferentspecies.BesidetheclassicalFirefly and Renilla luciferases,wealsoofferwiththenewGaussialuciferasesforimprovedsignalintensity:

ComparisonofdifferentspeciesluciferasesSpecies Luciferase Size QuantumYield Wavelenght ATP dependency SubstratePhotynus pyralis(Firefly) Fluc 550aa >88% 562nm YES D-luciferinRenilla reniformis(Seapansy) RLuc 311 aa >6% 480 nm NO coelenterazineGaussia princeps(Copepod) Gluc 185aa 1.6x1016Qps/mg 480 nm NO coelenterazine

Fireflyluciferaseiswidelyusedasareportergeneforstudyinggeneregulationandfunction,andforpharmaceuticalscreening.Itisaverysensitivegeneticreporterdue to the lackofanyendogenousactivity inmammaliancellsortissues. The Firefly luciferase is a 62 000 Dalton protein, which is activeasamonomeranddoesnotrequiresubsequentprocessingfor itsactivity.TheenzymecatalyzesATP-dependentD-luciferinoxidationbyoxygen intooxyluciferinwithemissionoflightcenteredon562nm(figure1).

However,thelightproductionresultingfromthereactionleadstoformationofsuicidaladenyloxyluciferinattheenzymesurface.Itresultsinveryshorthalf-lifeofthelightemissionwithaflash-typekinetics.Severalsubstanceshavebeendescribedtoprolonglightproductionbyregeneratingenzymethroughremovinginhibitoryoxyluciferinfromtheenzymesurface.Buttheduration(10-15min)isstilltooshortforbatchprocessscreening.

Ourluciferaseassaykitsprovidealonglastingsignal(steadyglow)bypreventingtheformationofadenyl-oxyluciferinattheenzymesurface.

Technical tip

Microplate Readers

InterchimandBerthold collaboration supports further yourworks.Many of our fluorescence andluminescencereagentsandkitswerevalidatedwithBertholdTechnologiesmicroplatereaders.

*MithrasLB940MultiModeReader. Includesavarietyoftechnologieswithsamplesinjectorsandrobotintegrationmodule.

. Variousformats(fromPetridishesto1536wellplates)

. Absorbance

. LuminescenceFlash&Glow

. Fluorescence

. topandbottommeasurement

. Polarisation (FP)

. FRET

. BRET

. AlphaScreen™

. TRF&HTRF®(TimeResolvedFluorescence&TimeResolvedFRET)

*CentroXSLB960LuminometerArobust,versatileandsensitivemicroplateluminometer(lowestcrosstalk)existsalsoinaClinicalversion(LB961).

*TwinkleLB970FluorometerReadingfromaboveandbelow,fromPetridishesto864wellmicroplates.IdealforsensitiveFRETassays

*ApolloLB912AbsorbanceReader96wellsmicroplatein340-800nmrange,with8-channels

Figure1:BioluminescentreactioncatalyzedbyFireflyluciferase.


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Firefly Luciferase

■FireflyLuciferase1-StepAssayKit,2hreading» Linearrange–Assaylinearoversevenorders of magnitude» Limitofdetection–lessthan1fgofluciferase per sample» Nodisposalproblemsorhazardsareassociatedwiththeuseofthisluciferaseassaykit» Reproducibility–CVlessthan5%

Luciferase 1-Step assay system is a homogeneous high sensitivity firefly lu-ciferasereportergeneassaykitwithahalf-lifeof2hoursforthequantificationoffireflyluciferaseexpressioninmammaliancells.Thiskitisspeciallydesignedforbatchprocessingsystemsusingmicroplatessuchas96-wellplates.Inad-dition,InterchimLuciferase1-Stepassaykitoffershighersensitivityandwiderdynamicrangefordetectingluciferaseactivitywithinmammaliancells,consis-tentreproducibilityandcosteffectivenessalongwiththeaddedconvenienceofa one step assay.

SensibilitystudyontheMithrasluminometerfromBertholdTechnologies:Different quantities of Firefly luciferase on the range of 0.0001-1 µg(0.005-50µg/ml)havebeenassayed.

Description P/N : QtyFireflyLuciferase1-StepAssayKit FP-BX0320 100ml(1000testsin96-wellplate)

FP-BX0321 100ml(1000testsin96-wellplate)

■FireflyLuciferaseStableAssayKit,3-5hreading» Simple : single step assay» Highsensitivity:highersensitivitythanotherssteadysubstrates» SuitableforHTS-batchprocessing

Thiskitisahomogeneoushighsensitivityfireflyluciferasereportergeneassaykitwithahalf-lifeof3-5hoursforthequantificationoffireflyluciferaseexpressioninmammaliancells.Itisspecificallydesignedforbatchprocessingsystemsusinghigh-densitymicroplatessuchas384-and1536-wellplates, in high throughput environments.

Description P/N : QtyFireflyLuciferaseStableAssayKit,3-5hreading FP-BU6870 10 ml

FP-BU6871 100 ml

Kitcontents(10/100/1000ml):1vial(2.5/25/250mg)ofD-Luciferin1bottle(10/100/1000mL)FireflyAssayBuffer10mlaresufficientfor100,400and3,300assaysin96-well,384-welland1536-wellmicroplates.

FP-BU6872 1000 ml

■FireflyLuciferase,recombinant,fromPhotinuspyralisLuciferasecanbeusedtodetecttraceamountsofATP(signallingbiologicalcontamination).LessthanorequaltoonefemtomoleofATPcanbedetectedusing0.2μgofluciferase.RecombinantFireflyluciferasecanalsobeusedtopreparestandardcurveofreportergenesforthestudyofgeneexpression.

Description P/N : QtyFireflyLuciferase,recombinant,fromPhotinus pyralis FP-D1826B 1 mg

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Firefly Luciferase

■MammalianExpressionVectors:pCMVLuxA&pCMVLuxBAsinglereportertomonitorexpressionoftwoclonedgenes

Thelight-emittingreactionofthemarinebioluminescentbacteriumVibrio harveyi iscatalyzedby thebacterial luciferaseenzymewhichexistsasanalpha-betaheterodimerencodedbytheluxAandluxBgeneswithsubunitmolecularweightsof 42Kand 37K respectively.The enzyme catalyzes a reactionwith FMNH2,oxygenandalong-chainaldehydeassubstratestoyieldvisiblelightat490nm.AnewluciferasemarkergenedetectionsystemhasbeendevelopedbaseduponthisbacterialluciferaseisolatedfromVibrio harveyi.Sequencesencodingthetwoluciferasesubunits,luxAandluxBhavebeenclonedintotwoseparatevectors.ThesevectorsalsoincludeaCMVpromoterforexpressioninmammaliancellsaswellasanampicillin resistancegene (100ug/mLampicillin resistance) forselectionandamplification,theSV40polyadenylationsequenceandtheSD/SA-RNAsplicedonorandacceptorsequenceformaximumexpression.Inaddition,theLuxAorLuxBgenecanbeexcisedusingtheflankingNotIsitestoallowtheinsertionofothergenestobeexpressedunderthesameregulatoryelementsinmammaliancells.Thesesystemsarebeingdevelopedtomonitorregulationofexpressionfortwoindependentvectorconstructs,uponthedualexpression.

Description P/N : QtypCMVLuxAMammalianLuxAExpressionVector DO8130 20µgpCMVLuxAMammalianLuxBExpressionVector DO8140 20µg

■FireflyluciferasesiRNAconstructs:siFLucsiFLucaresiRNAconstructsdesignedtoknockdownFireflyluciferase.Inourcontrolexperiments,thissiRNAcanknockdownFireflyluciferaseactivityby~80%.ThetargetsequencematchespGL3-control,andisdesignedtosilenceFireflyluciferaseexpressedbyaco-transfectedpGL3-controlvector.

Description P/N : QtypRNA-U6.1/Neo/siFLuc(positive control in mammalian transfection) BG9670 10µg

pRNA-U6.1/Neo/siFLucisasiRNAexpressionvectordesignedformammaliantransfection.ItusesU6promoterforsiRNAexpression.ThisvectorcontainssiRNAconstructforfireflyluciferase,andcanbeusedasapositivecontrol.ItcanalsobeusedasasiRNAvectorusingBamHIandHindIIIforsiRNAinsertion.

Pleasecontactusforotherspromotersavailable:» pGL:HEK293cellstransfectedwithpGL3-control(0.16ug)andpRL-TK(0.16ug)» U6-siLuc:HEK293cellstransfectedwithpGL3-control(0.16ug),pRL-TK(0.16ug), and1.6ugofpRNA-U6.1/Neo/siLuc» H1-siLuc:HEK293cellstransfectedwithpGL3-control(0.16ug),pRL-TK(0.16ug), and1.6ugofpRNA-H1.1/Neo/siLuc

U6:HEK293cellstransfectedwithpGL3-control(0.16ug)andpRL-TK(0.16ug),and1.6ugofpRNA-U6.1/Neoemptyvector.


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Firefly Luciferase

■pRNALuc/NeoformonitoringtranscriptionalactivityThepRNA-Luc/Neoincludesamodifiedcodingregionforfirefly(Photinus pyralis)luciferasethathasbeenoptimizedformonitoringtranscriptionalactivityintransfectedeukaryoticcells.ThepurposeofthisreportervectoristoscreenforefficientsiRNAforthetargetgeneusingLucactivityasareportergene.TheprincipleisthatwhenasiRNAsilencethetargetgenebydegradingmRNA,theLucwillnotbeexpressedeither,becausethemRNAforboththegeneandLucasawholemoleculeisdegraded.Theassayofthisgeneticreporterisrapid,sensitiveandquantitative.

Description P/N : QtypRNA-Luciferase-Neomycine DT3120 10µg

Relatedproductβ-Amyloid(1-40) HT8360 0.5mg

HT8361 1 mg

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Renilla Luciferase

■RenillaLuciferaseAssayKitReportergeneusedasnormalizingtransfectioncontrol

» SensitivityandLinearity:Linearcorrelationbetweenluciferasegeneexpressionandlightoutputfortransfectionusing1ngto1µgDNAofa Fireflyluciferasereporterconstruct» Convenientlypackagedsubstratesizespermittingyoutorunavariablenumberofassays

■RenillaMullereiLuciferase,recombinantDescription P/N : QtyRenillaMullereiLuciferase,95%purity(morethan8x1014Qps/mg) FP-BX6710 1 mg

■RenillaMullereiLuciferase,pUC19plasmid(pRLuc) Theseproteinusescoelenterazineandcoelenterazinederivativesassubstrate.TheRenillaproteinexpresseswellinbacteria.

Description P/N : QtypUC19pRLuc FP-BS8180 25µg

Renilla luciferase has been used as a reporter gene for studyinggene regulation and function in vitro and in vivo. Recently, Renillaluciferasehasbeenwidelyusedinmultiplextranscriptionalreporterassaysorasanormalizing transfectioncontrol forFirefly luciferaseassay. Renilla luciferase, a monomeric 36 000 Dalton protein,catalyzes coelenterazine oxidation by oxygen to produce light.The enzyme does not require post-translationalmodification for itsactivity,andmayfunctionasageneticreporterimmediatelyfollowingtranslation.nativeCoelenterazineisthenaturalsubstrateforRenillaluciferase. However, over a dozen of coelenterazine analogs havebeen synthesized, now commercially available from Interchim.Thesecoelenterazineanalogsall functionas substrates forRenillaluciferasewithdifferentproperties intermsofemissionwavelength,cellmembranepermeabilityandquantumefficiency.

Coelenterazinealsoemitslightfromenzyme-independentoxidation(autoluminescence),enhancedbysuperoxideanionandperoxynitriteincellsandtissues.Throughtheuseofaspeciallydesignedcoelenterazinederivativeandbufferformulation,theRenillaLuciferaseAssayKityieldsreliable,linearresultswithminimalautoluminescencebackgroundandsuperiorsensitivity.

Description P/N : QtyRenillaLuciferaseAssayKit FP-BE7930 150tests

FP-BE7931 1000 testsKitcontents:Coelenterazine,RenillaLuciferaseLysisBuffer,RenillaLuciferaseAssayBuffer,RenillaLuciferaseAssayEnhancer

Relatedsubstrates:See"Coelenterazines",asstandaloneproducts


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Firefly & Renilla Luciferase

■FireflyandRenillaLuciferasesAssayKit» SensitivityandLinearity:Linearcorrelationbetweenreportergeneexpressionandlightoutputfortransfectionusing1ngto1µgDNAof eitherFireflyorRenillaluciferasereporterconstruct.» LowAutoluminescence&HighSensitivity:ReducedautoluminescencebackgroundforRenillaluciferaseassayandconsequentlyincreased sensitivity. » Convenient:Onekitforbothluciferaseassays.

FireflyandRenillaluciferasesarewidelyusedasreportergenesfor studying gene regulation and function, and for pharmaceutical screening. Renilla Luciferase is often used in conjunction withFirefly Luciferase as a normalizing transfection control or formultiplextranscriptionalreporterassays.Aswithmanyenzymes,Firefly luciferaseandRenilla luciferase followMichaelis-Mentenkinetics and thus maximum light output is not achieved untilsubstrates (above the Km) and co-factor are present in largeexcess.Whenassayedundertheseconditions,lightemittedfromthe reaction is directly proportional to the number of luciferaseenzymemolecules.OurFirefly&Renilla luciferase assay kit isdesigned for detection and quantification of Firefly and Renillaluciferase reporter enzymes from cultured cells in a simple, efficientandlinearfashion.

Description P/N : QtyFireflyandRenillaLuciferasesAssayKit FP-BE7810 10 ml

FP-BE7811 100 mlKitcontentsper10ml:2x1mgD-Luciferin,100uL100XCoelenterazine,10mL5XPassiveLysisBuffer,10mLFireflyLuciferaseAssayBuffer,10mLRenillaLuciferaseAssayBuffer,10mLRenillaLuciferaseAssayEnhancer.

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Gaussia Luciferase

■HumanizedGaussiaLuciferaseGaussialuciferase,anovelreporterforgeneexpression,isthesmallestandbrightestknownluciferase.Recommendedforstudyingweakpromoters,hard-to-transfectcellsandHTSapplications.

» Greaterbrightness: Gaussialuciferaseexpressedinmammaliancellsisasmuchas750-foldbrighterthannativeRenilla reniformis luciferase» Avoidcelllysis:Gaussialuciferasewithsecretionsignalissecretedintothemedia.Itisthereforenecessarytoonlyassaysupernatantsfor enzymeactivitywithouttheneedforlysingthecells.Considerabletimeissavedsincetimecourseexperimentscanbeperformedsamplingthe samegroupoftransfectedcellswithoutlysingthecell» Extremelystabletoelevatedtemperature:upto60°Candapprox.20%recoveryfollowinga15minuteincubationat99°C» pH resistance : surviving a pHrangeof3-11» Resistancetodetergents:1-5%non-ionicdetergents(NP-40,TritonX-100,TritonX-114,CHAPSO),cholate,deoxycholateetc» Abilitytorecoveractivityaftertreatmentwith7Mguanidinechlorideor8Murea+NP-40

Gaussia luciferase uses coelenterazine and its derivatives to catalyse the oxidativedecarboxylationofcoelenterazinetoproducecoelenteramideandlight.Ithasanemissionspectralpeakat480nm.

Thespecificactivityofthisluciferaseinthepresenceofhighconcentrationsofcoelenterazine(10μM)isextremelyhigh:1.24x1016Qps/mg(Quantapersecond per milligram)

Description P/N : QtypGluc-basic-1promoter-lesswithsecretionsignal BU2550 25µgApromoterlessvectorwithaMCSsiteupstreamofthehumanizedGaussialuciferasecodingsequence(withsecretionsignal).ThisvectorisdesignedforpromoteranalysisandwillexpresssecretedGaussialuciferase.Thetransfectedcellscanbereusedformultiplesampling.

pCMV-Gluc-1positivecontrolwithsecretionsignal BS8160 25µgThispositivecontrolvectorisveryusefulinevaluatingtheefficiencyoftransgeneexpressionusingGaussialuciferaseasareporter.ThisvectorhasbothAmpicillinresistanceandNeomycinresistance.ThereforeitcanbeeasilypropagatedinE. coliandcanbeusedtoestablishstablecelllinesexpressingGaussialuciferase.

pCMV-KDEL-Basic-1forintracellularexpression BU2570 25µg

pCMV-KDEL-Gluc-1forintracellularexpression BU2560 25µg

Relatedproducts:Anti-GaussiaLuciferase,rabbittiter>1:10000 CJ3430 250µlUptiFectin-Ontransfectionreagent CK5060 0.5ml

■GaussiaLuciferaseAssaykitThe GaussialuciferaseassaykitstabilizestheflashsignalemittedbytheGaussialuciferasethusmakingitpossibletouseitasareportergenefor high throughput applications

The humanized Gaussia luciferase is secreted into the culture media and only the media needs to be assayed by the addition of nativecoelenterazine.

The GaussiaAssayReagent(GAR)ispreparedfreshlybydilutingthecoelenterazinestockwiththeassaybuffer.Theassayisperformedasfollowing:-Add50µlofGARto20µlGaussialuciferasesamplefrommicrotiterorculturewellsamples-Mixwellandreadinluminometer

Description P/N : QtyGaussiaLuciferaseAssaykit FP-BY7160 5ml(100tests)

FP-BY7161 50ml(1000tests)Kitcontains:pre-dissolvedcoelenterazine(100Xconcentration)andanassaybufferwithstabilizers(increaseemissionupto45minutes).


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Coelenterazines

Coelenterazine–coelenterateluciferin–isthesubstrateforanumberofmarinebioluminescentenzymes,includingthosefrommarineorganismsRenilla, Gaussia, Pleuromamma (luciferases) Aequorea (aequorin) and Obelia (obelin).Insomeofthesereactionsitisutilizedasasimplesubstratebeingcatalyticallyturnedoverinthebioluminescentreactioncatalyzed by luciferases),whileinothers,suchasaequorinorobelin,itisincorporatedas part of the photoprotein.

■Coelenterazine,nativeThe native coelenterazine, the luminophore of the native aequorin complex, is the standard substrateusedinmanyapplicationsusingluciferasereporter assays. Bioluminescent detection of calcium concentration is highly sensitive in a broadconcentrationrange(0.1μMto>100μM)1-4.Monitoringof reporter genes (phot gene and luc gene) using coelenterazineisalsoamajorapplication.Otherusesofcoelenterazineincludebioluminescenceresonanceenergy transfert (BRET)5,10 and chemiluminescent detection of superoxide anion and peroxynitrite in cells or tissues 6-9.Coelenterazine native is recommendedwhena fastregeneration is important.

References1)Meth.CellBiol.40,305(1994);2)Meth.Enzymmol.172,164,(1989);3)J.BioChem.105,473(1989);4)J.Chem.Soc.Chem.Commun.21,1566(1986)5)Meth.Enzymol.57,271(1978);6)TetrahedronLett.31,2963(1973);7)Nature256,236(1975);8)Anal.BioChem.219,169(1994);9)proc.Natl.Acad.Sci.USA96,151(1999);10)MolecularPharmacology,70:1802-1811(2006)

Description P/N : QtyCoelenterazine,native UP972331 50µgHighest purity FP-97233B 250µg

UP972333 1 mgUP972334 10 mg

Pleasecontactusforbulkquoteatinterbiotech@interchim.comAlsoavailable:Coelenterazinenativeforinvivoapplications#FP-BV0730.

ThisresultpromptstoprepareFRESHCoelenterazineworkingsolutionandthentoletitsitfor15-20minutesatroomtemperaturebeforeuseinordertoachievebestaccurateandhighsensitivity.

■Coelenterazine400aProteininteractionsstudyinBRETwithGFPacceptor

Colenterazine400a,alsoknownasDeepBlue™C,isacoelenterazinederivativethatservesasasubstrateforRenilla luciferase(Rluc)andgeneratesanemissionpeakcenteredaround400nm.ItisthepreferredRlucsubstrateforBRETstudiesbecauseithasminimalinterferencewiththeemissionoftheGFPacceptor(GFPvectorsarepresentedintheBioscienceInnovationcatalog).

Description P/N : QtyCoelenterazine400a UPBB8391 50µg

FP-BB839B 250µgUPBB8392 1 mg

SeeothercoelenterazinesintheBioscienceInnovationcatalog.

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Coelenterazines

■CoelenterazineH(Benzyl-Coelenterazine)ForcalciumassayinvitroorproteininteractionsstudyinBRETwithYFPacceptor

Coelenterazine H works better with Calcium activated photoproteins (Aequorin, obelin) compared to native Coelenterazine; however thisis true only in vitro. This cell membrane-permeable, very sensitive, specific, intracellular luminophore is useful for measuring changesin Ca2+ i.e. in cells that have been transfected with apoaequorin cDNA. In this system, coelenterazine is required for the regeneration ofaequorin, a protein that emits light in the presence of calcium, fromapoaequorin produced in cells.The luminescence intensity appears to bedirectly proportional to the Ca2+ concentration. Coelentrazine-H exhibits an approximate 16-fold greater luminescence intensity (emission max.:~466nm;half-timetotalof0.6-1.2sec.)ascomparedtothenativeCoelenterazine.HasbeenusedtomeasureintracellularCa2+ signals in Dictyostelium discoideumchemotaxisandinplantwoundhealing.

Thebioluminescenceresonanceenergytransfer(BRET)method,betweenRenilla luciferaseandavariantofGFP,theyellowfluorescentprotein(YFP)allowsreal-timedetectionofprotein-proteininteractionsinvivo.

Description P/N : QtyCoelenterazineH UPR30782 50µg

FP-R3078B 250µgUPR30783 1 mgUPR30784 10 mg

Relatedproduct:BAPTA,AM FP-486103 25mg

FP-486104 20x1mgThisCa+2chelatorthatdelaysthepeakandincreasesthedurationoflightemissionfromaequorin

Alsoavailable:CoelenterazineHforinvivo#FP-BV0680

■OthercoelenterazinesavailableTableofLuminescentPropertiesofCoelenterazineProductswithApoaequorin*SeecompletedescriptionsintheBioscienceInnovationcatalog.

Coelenterazine P/N : R1(2) R2(6) R3(8) λ max. RelativeLuminescence Relative Half-rise# # # Emission(nm) capacité § Intensity § Time(s)§

CoelenterazineNative FP-97233A OH OH Phe 465 1.00 1.00 0.4-0.8Coelenterazinecp FP-R3079A OH OH CP 442 0.95 20 0.15-0.3Coelenterazinef FP-43876A F OH Phe 473 0.80 18 0.4-0.8Coelenterazinefcp FP-R4711A F OH CP 452 0.57 135 0.4-0.8Coelenterazineh FP-R3078A H OH Phe 464 0.82 10 0.4-0.8Coelenterazinehcp FP-08353A H OH CP 444 0.67 190 0.15-0.3Coelenterazinei FP-R3080A I OH Phe 476 0.70 0.03 8Coelenterazineip FP-R4712A I OH 2P 441 0.54 47 1Coelenterazinen FP-39819A Naph OH Phe 467 0.26 0.01 5

*AlldatasfromBioChem.J.261,913(1989)(otherdatacanbefoundinO.ShimorauraCellCalcium14,373(1993)§ Luminescence capacity is the total light emission of aequorin in saturatingCa2+. Intensity of luminescence insaturating Ca2+ measured at max emission wavelength. Half-rise time is the delay elapsed to get 50% of themaximum emission.# substituant groups R1, R2 and R3, in positions 2, 6 and 8, are hydrogen (H), hydroxyl (OH), Phenyl (Phe),CycloPentyl(CP),2-propionyl(2P),Napthyl(Naph),methyl(Met).Coelenterazineehasa-CH2CHbridgebetweenthe6-phenyl-OHandposition2oftheimidazopyrazinonecore.


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D-Luciferins

Thepopularreportergene,lucgene,encodestheFireflyluciferase.TheATP-dependentoxidationofthesubstrateD-luciferinbyoxygenproducesanemissioncenteredaround562nm.ThelightoutputisproportionaltoluciferaseconcentrationwhenbothD-luciferinandATPexistinlargeexcess.

InterchimsuppliesD-luciferininvariousforms:freeacid,potassiumsaltandsodiumsaltandderivativeswithacetoxymethyl(AM),methyletherandDMNPE.Thepotassiumandsodiumsaltformsarethemostpopularbecausetheyarereadilywater-soluble.Thepotassiumsaltisalsotheformusedinliveanimalassay.Interchim’sD-Luciferinsarestrictlycontrolledviaseveralchemicalanalysesandalsoviaafinalenzymeassaytoensureconsistency.

Description P/N : QtyD-Luciferin,freeacid,>99,0% FP-27060A 25mg

FP-27060B 100 mgFP-27060C 250mgFP-27060D 1 g

D-Luciferin,K+ salt, >99,0% FP-M1224A 25mgPotassium salt is the recommand salt form for in vivo uses. FP-M1224B 50mg

FP-M1224C 500mgFP-M1224D 1 g

D-Luciferin,Na2+ salt, >99,0% FP-726045 10 mg FP-72604A 25mgFP-72604B 50mgFP-72604C 1 g

D-LuciferinAM,cellpermeant FP-M1909A 5mgThecell-permeantD-luciferinAMesterenterseasilyintolivecells,andiswellretainedonceitiscleavedbyintracellularesterasestoD-luciferin.

D-Luciferinethylether FP-CF4421 10 mgCellpermeantanalogwith30%highersignalintensity

DMNPE-cagedD-Luciferin FP-21639A 5mgDMNPE-cagedD-luciferinisaD-luciferinesterderivativewhichcancrosscellmembranesefficiently.Onceinsidethecells,theesteriscontinuouslyhydrolyzedtoasupplyofD-luciferin.AlternativelyaburstofD-luciferinisgeneratedbyUVphotolysis.References:Luque-OrtegaJ.R.etal.-InVivoMonitoringofIntracellularATPLevelsinLeishmaniadonovaniPromastigotesasaRapidMethodToScreenDrugsTargetingBioenergeticMetabolism,AntimicrobialAgentsandChemotherapy,p.1121-1125,Vol.45,No.4(2001)

D-Luciferin6-methylether,Nasalt FP-M1418A 10 mgD-Luciferinmethyletherhasbeenproposedtobeasubstrateformicrosomaldealkylase/cytochromeP450.DemethylationofthesubstrategeneratesD-luciferin,andthuscanbedetectedviabioluminescencewithextremelyhighsensitivity.Reference:DenburgJ.etal.Substrate-bindingpropertiesoffireflyluciferaseI.Luciferin-bindingsite.ArchsBiochem.Biophys.134,381-394(1969).

D-Luciferin-6-0-β-D-galactopyranoside FP-CQ6410 5mgβ-GalactosidasesubstrateReference:Yang,Y.etal.,Homogeneousenzymeimmunoassaymodifiedforapplicationtoluminescence-basedbiosensors,Anal.Biochem33:102-107(2005)

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Substrates for glucosidases reporter systems (β-galactosidase, β-glucuronidase, β-glucosidase)

■β-GalactosidasefluorescentsubstratessamplerKitThiskitconsistsofsamplesofseveralofourmostpopulargalactosidasesubstratesandtheirreferencefluorophoresallowingmultiplexedanalysisoflacZβ-Galactosidaseactivityatavarietyofwavelengths.Thiskitisperfectforthoseoccasionswherethepreferredwavelengthofdetectionisunderdevelopment.

Description P/N : Qtyβ-GalactosidasesubstratessamplerKit FP-BM8400 1kitContains:Subst.FDG #52476A,10mgFluo.Std.Fluorescein #193659,10mgSubst.Res-Gal #524739,10mgFluo.Std.Resorufin #95432A,10mgSubst.TFMU-Gal #M11419,10mgFluo.Std.TFMU #434769,10mg

■OthercolorimetricsubstratesforglycosidasesDescription P/N : Qtyo-NPG(o-Nitrophenyl-β-D-galactopyranoside,MW:301.3;λabs(cleaved):420nm) UP556683 5gX-GLU(5-Bromo-4-Chloro-3-Indolyl-B-D-Glucopyranoside,MW:408.6) UP193325 100 mgX-GAL(5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside,MW:408.6) UP40534M 1 g

Eachsubstrateisavailableseparately,andalsomanyotherderivatives(inhibitors,activators,...).Pleaseinquire.


Enzym

es Detection


Alkaline phosphatase assays

Phosphatasesareaspecificclassofenzymesthatcatalyzetheremovalofphosphategroupsfromproteins,whereaskinasesenzymaticallyaddaphosphatetoaprotein.Phosphatasesareimportantinsignaltransductionbecausetheyregulatetheproteinstheyareattachedto.Toreversetheregulatoryeffect,thephosphatehastoberemoved.Cellproliferationanddifferentiationareregulatedbyphosphatases.

Phosphatasesservealsoasenzymemarkers,allowingtoquantifyphosphataseactivityindifferenttypesofcells.AlkalinephosphataseisfinallyahighlysensitiveenzymeforELISA,immuno-histochemical,Northern,SouthernandWesternblotapplications.

■FluorimetricAssaykitsTheAlkalinePhosphataseAssayKitsusefluorogenicphosphatasesubstrate,toquantifyalkalinephosphataseactivityinsolutions,incellextracts,inlivecellsaswellasonsolidsurfaces(suchasPVDFmembranes).Thekitsprovidealltheessentialcomponentswithouroptimized"mixandread"assayprotocolthatiscompatiblewithHTSliquidhandlinginstruments.

■AlkalinePhosphataseAssayKit,BlueFluorescenceSubstrate:MUPλex./em.:360/449nmSensitivity:0.3pgofalkalinephosphatase

Description P/N : QtyFluorimetricAlkalinePhosphataseAssayKit,Bluefluorescence

JQ6730 500tests

■AlkalinePhosphataseAssayKit,GreenFluorescenceSubstrate:FDP.λex./em.:490/514nmSensitivity:0.1pgofalkalinephosphatase

Description P/N : QtyFluorimetricAlkalinePhosphataseAssayKit,Greenfluorescence

JQ6740 500tests

■AlkalinePhosphataseAssayKit,RedFluorescenceSubstrate:Phospholite™Redλex./em.:570/590nmSensitivity:0.2pgofalkalinephosphatase

Description P/N : QtyFluorimetricAlkalinePhosphataseAssayKit,Redfluorescence

JQ6750 500tests

Alkalinephosphatasedoseresponseon96-wellblackplatewith30minutesincubationtime(n=3).



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Alkaline phosphatase assays

LuminometricAPAssaykit

■LuminometricAlkalinePhosphataseAssayKitSubstrate:proprietaryluminescentsubstrateλem. : 560nmSensitivity:0.01pgofalkalinephosphatase

This Alkaline Phosphatase Assay Kit uses a luminogenicphosphatase substrate, to quantify alkaline phosphataseactivityinsolutions,incellextracts,inlivecellsaswellasonsolidsurfaces(suchasPVDFmembranes).Thisproprietaryphosphatase substrate generates a luminescent productthat produces strong luminescence upon interaction withphosphatase.

Description P/N : QtyLuminometricAlkalinePhosphataseAssayKit JQ6760 200assays

ColorimetricAPAssaykitsSeeAPcolorimetricassayssubstratesandkitsinImmunoassayreagentssection.

Acide phosphatase assays

■MUPPlusAlthoughMUPiswidelyusedfordetectingphosphatasesinsolutionitisnotwellsuitedforlivingcellorcontinuousassayssince4-methylumbelliferone,theenzymaticproduct,whichonlydevelopsmaximumfluorescenceatpHvalueof>10.Thus it isalsodifficult touseMUP for thedetectionofphosphatasesthathaveacidicoptimalpHrangesuchasacidphosphatases.FluoProbesispleasedtoofferMUPPlusthatisdevelopedtoaddressthispHlimitationassociatedwithMUPsubstrates.MUPexhibitsmaximumfluorescenceabovepH7.0,thusMUPPlussubstratecanbewellusedforcontinuousassays.ItcanalsobeusedfortheassaysthatrequireacidicpHsuchasacidphosphatases.

Description P/N : QtyMUPPlus™,sodiumsalt FP-JQ6710 25mgλex./em.:360/450nmMW:300g.mol-1



Enzym

es Detection


Protease Assay Kits

■ProteaseAssaysKits,Green&RedFluorescence» OptimizedPerformance:Optimalconditionsforthedetectionofgenericproteaseactivity» HighSpeed:Minimalhands-ontime» AssuredReliability:Detailedprotocolandreferencesareprovided

TheProteaseAssayKitsarewidelyusedfordetectionofgenericproteaseactivities.Thekitsuseacaseinderivativethat isheavily labeledwithgreenorredfluorescence,resultinginalmosttotalquenchingoftheconjugate'sfluorescence.Protease-catalyzedhydrolysisrelievesthisquenchingconjugate,yieldingbrightlyfluorescentdye-labeledpeptides.Theincreaseinfluorescenceintensityisdirectlyproportionaltoproteaseactivity.Theproteaseassaykitsdonotrequireanyseparationstepsandcanbeusedtocontinuouslymeasurethekineticsofavarietyofexopeptidasesandendopeptidases.

Thekitscontains: •Fluorescentlabeledcaseinwithhighratioofdye/protein(pH-insensitive) •Trypsin(aspositivecontrol) •Assaybuffer •Adetailedprotocol

Description P/N : QtyProteaseAssayKit,GreenFluorescence(488/520nm) BK962A 500tests*ProteaseAssayKit,RedFluorescence(546/575nm) BK963A 500tests*

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Lipase assays and Lipid study

■LipaseAssayKitFast and easy measurement of lipase activity in vitro, in cell preparations or in vivo

Lipasesareafamilyofenzymesthatreleasefattyacidsfromtriacylglycerolsinasitespecificmanner. Most lipases have optimum activityfor the primary ester groups of triglycerides, while some lipases remove fatty acyl groupsfromeithertheC-1orC-3acylpositions.Thesubstrateistypicallynotasinglemolecule,butanonaqueousphaseofaggregatedlipid.Lipaseactivity,ubiquitousamongmostcells, canbemonitoredusingthenewfluorescentsubstrate1,2-dioleoyl-3-pyrenyldecanoyl-rac-glycerol(Product # FP-M14031, λexc./em.: 342/470 nm)contained in the kit. Upon cleavage, thefluorescent fatty acid pyrenedecanoic acid(Product#FP-37853A,λexc./em.:341/377nm)isreleased and activity measurements are easily obtainedeitherinvitro,incellpreparations,orinvivo.Thekitcontainsenoughsubstratefornumerous assays and control experiments, and also contains reference standards and a detailed protocol for use.

References:HowardG.T.,etal."Sensitiveplateassayforscreeninganddetectionofbacterialpolyurethanaseactivity".Lett. Appl. Microbiol.32(3):211-4(2001)KoukerG.andJaegerK.E.,"Specificandsensitiveplateassayforbacteriallipases".Appl Environ Microbiol53(1):211-3(1987)

Description P/N : QtyFluorescentLipaseAssayKit BG8440 1kit(72assaysin96wellplate)

■UnboundfreefattyacidsAssay-ADIFABTheassaycanbeusedinavarietyofbiochemicalandclinicalapplicationsincludingthedeterminationof lipaseactivity, fattyacidbinding tomembranesandproteins,andserumunbound free fattyacid(FFAu)levels.ADIFABandADIFAB2areidealalsofordrugscreening.Theyareparticularlywell-suitedfordrugsthataffectcellularprocessesaswellasthoseinvolvingpurifiedenzymes.FFAu probes have been validated for high throughput assay. This includes robotic dispensing ofreagents,fluorescencescreeningandthedeterminationofFFAulevelsin96and384wellplates.ThissystemcanbeuseddirectlytoscreenfordrugsthataltercellularmetabolisminvolvingFFAorthatalterthebehaviorofenzymesthateitherproduceoruseFFA.ADIFAB2isahighaffinityversionoftheoriginalADIFABprobe.ItisformedbyacrylodanlabelingtheLeu72toAlamutantoftheIntestinalFattyAcidBindingProtein.ADIFAB2,similarlytoADIFAB,canbeusedtoassayunboundfreefattyacidlevelsbutprovidesgreatersensitivityforlowconcentrationsoftheFFAlevels.Forthoseconcentrationsbelowabout400nM,theincreaseintheADIFAB2emissionratioisabouttwicethatforADIFAB.ThefluorescenceofADIFABismeasuredwiththeratio505/432uponexcitationat386nm.ADIFAB2fluorescenceemissionspectraoccuratlongerwavelengths(550/457nmwithexcitationat375nm).BindingaffinitiesforADIFAB2areapproximatelytentimesgreaterthanforADIFAB.Ontheotherhand,ADIFABhasawiderrangeofsensitivitythanADIFAB2andispreferableforhigherconcentrationsofunboundfreefattyacids.

IntracellularFFAulevelsmeasuredwithADIFABmicro-injectedintofatcells–CopyrightFFAbiosciences

Description P/N : QtyADIFAB 040791 200µg

0470792 1 mg

ADIFAB2 BB6681 200µgBB6682 1 mg


Enzym

es Detection


Collagen and elastin study

Sensitivity:40ng of lysyl oxidase in solution

Lysyloxidaseisanextracellularenzymethatcatalyzesformationofaldehydes from lysine residues in collagen and elastin precursors. These aldehydes are highly reactive, and undergo spontaneous chemical reactions with other lysyl oxidase-derived aldehyderesidues,orwithunmodifiedlysineresidues.Thisresultsincross-linkingcollagenandelastinwhich isessential forstabilizationofcollagenfibrilsandfortheintegrityandelasticityofmatureelastin.Lysyloxidasehasbeenidentifiedasapossibletumorsuppressor.Lysyl oxidase activity in biological samples is traditionally andmostreliablyassessedbytritiumreleaseend-pointassaysusingradiolabeled collagen or elastin substrates involving laboriousvacuum distillation of the released tritiatedwater. This kit offersasensitivefluorescentassayforlysyloxidaseactivitythatutilizes1,5-diaminopentaneassubstrate,andreleasedhydrogenperoxideis detected using our HRP substrate in HRP-coupled reactions.Thismethodallowsthedetectionofsubng/mLlysyloxidaseandismuchmoresensitivethanthecurrentlyavailablefluorimetricassayfor this enzyme activity. This method eliminates the interference thatoccursinsomebiologicalsamplesandcanbereadilyusedtodetect lysyl oxidase activity in cell culture experiments.

Lysyloxidasedoseresponseon96-wellblackplatewith30minutesincubationtime(n=3).Theinsertshowsthelowlevelsoflysyloxidasedetection.

■LysylOxidaseAssayKit,RedFluorescence

Description P/N : QtyLysylOxidaseAssayKit,redfluorescence JQ7270 500assays

■CellulaseAssayKit,RedFluorescenceSubstrate:ResorufinCellobiosideReactionvolume:100µlλex./em. : 571/585nm

Cellulases are a family of enzymes that includeß-Glucosidases, endoglucanases, and exoglucanases. Theseenzymescleave theß-1,4-D-glycosidicbondsthat link the glucose units comprising cellulose. Inadditiontobeingproducedbyplants,cellulaseactivityis found inmany fungi andbacteria, including someplant pathogens. Most animal cells are not knownto produce cellulase; cellulolytic activity is oftencarriedoutinanimalsbysymbionts.However,recentevidence does suggest cellulase production in some animals, such as insects and arthopods. The study of cellulase activity has many applications in plant molecularbiology,agriculture,andmanufacturing.Cellulaseisalsobecomingimportantinthedevelopmentofalternativefuelsources,asglucoseobtainedfromcellulosehydrolysisiseasilyfermentedintoethanol.Activityofmostcellulasescanbemonitoredusingourlongwavelengthfluorescentsubstrate,ResorufinCellobioside,containedinthekit.Uponcleavage,thefluorescentcompound,Resorufin,isreleasedandactivitymeasurementsareeasilyobtainedinamicrotiterplatebasedassayformat.

SuspensionoffloweringbudsfromtwomatureArabidopsis thalianaplantsintriplicate(50μL)ona96-wellclear,flatbottomplatereadat3-minuteintervalsfor120minutes.

Description P/N : QtyFluorescentCellulaseAssayKit DO8110 200assaysKitcontains:SubstrateReagent,ReferenceStandard,ReactionBuffer,StopBuffer,DMSO

Cellulase Detection

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Acethylcholinesterase Detection

■aCella–AcetylcholinesteraseAssayBioluminescenceassayforMonitoringAcetylCholinEsteraseActivity

» FAST:Resultsin30seconds-5minutes» Homogenous : One-step,nowashassay» UltraSensitiveassaytomonitorAChEactivity» Versatile:Nervegas,pesticidemonitoring;drugscreeningapplications

Acetylcholinesterase (AChE) is one of themost important enzymes involved in nervetransmission.Theenzymeisboundtocellularmembranesofexcitabletissue(synapticjunction, endoplasmic reticulum, etc). Acute toxicity to humans and animals throughinhibitionofAChEbybothnervegasesandanimportantclassofpesticideshaslongbeenafieldofintensivescientificinvestigation.AChEinhibitorshavealsobeenusedclinicallyasAlzheimer’s treatments (e.g., tacrine (tetrahydroaminoacridine))andare thesubjectof increasing interest in variousdiseaseprocessesand treatment strategies.However,bothenvironmentaldetectionofAChEinhibitorsanddevelopmentofmodulatorsofAChEenzymaticactivityasdrugshavebeenhamperedbythedifficultyandcomplexityofthecurrent assay methods.

Assay Principle

ReactionI:AChE+Inhibitor NoAcetateandCholine.

ReactionII:Coupledenzymereaction+ATP Reactiondoesnotproceed.

ReactionIII:ATP(remaining)+Luciferase/Luciferin LIGHT

Tacrine (a mixed-mode inhibitor of AChE) was seriallydiluted in DI water. Next 10 μL of the dilutedTacrine (xaxislabelingrepresentsμMfinalconcentrationofTacrine)wasaddedtoawhiteopaque96wellmicroplatealongwith 50μLofcomponentA(AChEenzyme).Thesampleswereincubated for5minutesafterwhich50μLofcomponentBwasaddedtoallthewells.Datawascollectedusingaluminometer.DatashownrepresentsT=2minutesaftertheaddition of component B.

Description P/N : QtyaCella–AchEAssay CA6650 100 tests

CA6651 500testsCA6652 1000 tests

KitContents:Acetylcholinesterase,Detectionreagent,acetylcholineandcoupledenzymereaction,Controltomeasuremaximumluminescence


Calcium

Assays


Calcium Assays

IntracellularCa++levelshavebecomeimportantindicatorsfortheactivationstateofionchannelsandG-proteincoupledreceptorsaswellasforthephasesofapoptosisandcellinjury.ThoughtherespectivekineticsandtheabsoluteamountsoftheCalciumlevelsaredifferentforeachofthesephysiologicalprocessestherearecommonwaysformonitoringthem.LuminescentlabelslikeAequorinaswellasfluorescentonesareversatileandwidelyusedsolutionsformicroplateassays.Fura2andIndo-1provideratiometricreadouttherebyreducingeffectscausedbyleakingorbleacheddyes or varying assay conditions.

Productname MW(g·mol-1) λexc\λem max. (a)

Free Ca2+(nm)λexc\λem max. (a)HighCa2+(nm) Kd Ca2+(nM) Applications

Fluo-3AM(b) 1129 503/weak 505/526 390 .Mostclassicalcalciumindicator

Fluo-8AM 1000 490/weak 490/514 389.NoWashstepneeded.4timesbrighterthanFluo-3.Loadingatroomtemperature

Fluo-8HAM 1100 490/weak 490/514 232 .HighCa2+ concentration indicator

Fluo-8LAM 1100 490/weak 490/514 1860 .LowCa2+ concentration indicator

Rhod-4AM 530/weak 530/555 525.Redcalciumindicator.4timesbrighterand10timeslargerwindowsassaythanRhod-2.Loadingatroomtemperature

Fura-PE3AM 1258 335/495 380/495 250.LeakageresistantformofFura-2.Ratioofreadswith2differentλex..Avoidsinterferenceduetodyedistributionandphotobleaching

Indo-PE3AM 1266 338/480 338/410 260.LeakageresistantformofIndo-1.Ratioofreadswith2differentλem..Avoidsinterferenceduetodyedistributionandphotobleaching

(a) after hydrolysis(b)AM esteraremembrane-permeantandthusincreasesgreatlycellloadingthatcanbeperformedbysimpleincubationofthecellsortissuepreparationinabuffercontainingtheAMester.Pluronic®F-127,amildnon-ionicdetergent,canfacilitateAMestersloading.TheAMestersthemselvesdonotbindtoCa2+.However,oncetheyhaveenteredthecells,theyarerapidlyhydrolyzedbyintracellularesterasesintotheparentCa2+compounds,thusbecomingfullyfluorescentuponbindingtoCa2+.ManyotherionindicatorsareavailableinourInterchim’rangeofdye.Pleasecontactusforspecificapplicationneeds.

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Single λexc./em. Ca2+ indicators

■Fluo-3AMStandardCa2+ concentration indicator

» Largedynamicrange» Lowcompartmentalization» AppropriateapparentCa2+ bindingaffinity

Fluo-3 has an absorption spectrum compatible withexcitation at 488 nm by argon-ion laser sources, and a>100 fold fluorescence intensity increase in responseto Ca2+ binding. Fluo-3 proves to be the generally mostapplicableCa2+ indicator, even if it ismore susceptible tophotobleachingthanmanyoftheotherCa2+ indicators.

Description P/N : QtyFluo-3AM FP-78932A 1 mgλexc./λem.=505/523nm FP-R1245A 1mgFluoProbesPureGradeKd=390nm FP-78932B 10x100µg

FP-78932C 20x50µgFP-M2036A 1ml(1mMsolutioninDMSO)FP-78932D 50mg

■Fluo-8AM-NoWashThenextgenerationcalciumindicatorforautomatedscreening(HTS)applications

Fluo-3 Fluo-4 Fluo-8

» Increasedsignalintensity» Rapiddyeloading:dyeloadingatRT(ratherthan37°CrequiredforFluo-4AM)» Convenientandrobust:Nowashstepneeded.» Performedin96or384-wellmicrotiter-plate

The Fluo-8NW(NoWash)cancrosscellmembrane.Onceinsidethecell,thelipophilicblockinggroupsofFluo-8NWarecleavedbyesterases,resultinginanegativelychargedfluorescentdyethatstays insidecellsand itsfluorescence isgreatlyenhanceduponbindingtocalcium.Whencellsstimulatedwithagonists, thereceptorsignalsthereleaseof intracellularcalcium,whichgreatlyincreasethefluorescenceofFluo-8NW.Thecharacteristicsofitslongwavelength(490/514 nm), high sensitivity, and >100 times fluorescence enhancement (when it forms acomplexwithcalcium)makeFluo-8NWanidealindicatorformeasurementofcellularcalcium.

ATPdoseresponsesinHEK-293cellsDescription P/N : QtyFluo-8NoWashCalciumAssayKit,Mediumremoval1 CJ2560 10 plates

CJ2561 100 plates

Fluo-8NoWashCalciumAssayKit,1%FBSMedium2 CJ2550 10 platesCJ2551 100 plates

Fluo-8AM CP7501 5x50µgλexc./λem.:(Hydr.,Ca2+):505/523nm;Kd(Ca2+)=390nM;MW:1000

Relatedproducts:Probenecid,Cellculturetested,tosuppresseffluxofdyes FP-288652 10x150mgProbenecid,watersoluble FP-288653 10x150mgIonomycin,Ca2+ ionophore FP-53989A 1 mgIonomycin,Ca2+ ionophore FP-53989B 5mg

Note1 : It is important to remove thegrowthmediumin order to minimize background fluorescence, andcompoundinterferencewithserumorculturemedia.Note2:Alternatively,onecangrowthecellsingrowthmediumwith0.5-to1%FBStoavoidmediumremovalstep.

Calcium

Assays



■Fluo-8HAMHighcytosolicCa2+ concentration indicator

HighCa2+concentrations,presentinsomeorganelles(mitochondria,vacuoles)andinexcitablecells(fibroblasti.e.),werehardlydetected:standarddyesFluo-3,Fluo-4andRhod-2havetoohighaffinityforCa2+.

Themeasurement of cytosolic freeCa2+ ion concentrationwith lowaffinityCa2+ indicatorshasadvantages for kinetic studiesof cytosolic [Ca2+] transientswhencomparedwithmorecommonlyusedhighaffinityCa2+indicators.Theirdynamicrangeandlinearityarebettersuitedtomeasurementofhigh-localisedtransientconcentrationchangesthatexistnearsitesofinfluxorrelease,andtheadditionalbufferingintroducedbytheindicatorisminimised.

Description P/N : QtyFluo-8HAM(490/514nm) FP-CP7531 10x50µgλexc./λem.:(Hydr.,Ca2+):490/514nm;Kd(Ca2+)=232nM;MW:1100 FP-CP7530 1 mg

RelatedproductsMag-Fura-2AM(λexc./λem.:369,329/510nm) FP-35374C 20x50µg

■Fluo-8LAMLowcytosolicCa2+ concentration indicator

Description P/N : QtyFluo-8LAM FP-CP7551 10x50µgλexc./λem.:(Hydr.,Ca2+):490/514nm;Kd(Ca2+)=1.86µM FP-CP7550 1 mg

Cal

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Ass

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Rhod-4™ is the brightest red calcium indicator available for HTSscreening. Once inside the cell, the lipophilic blocking groups ofRhod-4™ are cleaved by non-specific cell esterase, resulting in anegatively charged fluorescent dye that stays inside cells, and itsfluorescenceisgreatlyenhanceduponbindingtocalcium.Whencellsstimulatedwithscreeningcompounds, thereceptorsignals releaseof intracellular calcium,which greatly increase the fluorescence ofRhod-4.Thecharacteristicsof its longwavelength,highsensitivity,and >250 times fluorescence increases (when it forms complexeswith calcium) make Rhod-4™ an ideal indicator for measurementofcellularcalcium.ThisRhod-4NWCalciumAssayKitprovidesanoptimized assay method for monitoring G-protein-coupled receptors (GPCRs)andcalciumchannels.Theassaycanbeperformed inaconvenient 96-well or 384-well microtiter-plate format and easilyadapted to automation.

The Rhod-4 NW Calcium Assay Kit provides a homogeneous fluorescence-based assay for detecting the intracellular calciummobilization,andapreferredmethodindrugdiscoveryforscreening.Cells expressing a GPCR of interest that signals through calciumarepre-loadedwithourproprietaryRhod-4NWwhichcancrosscellmembrane.

Figure1.CarbacholDoseResponseinHEK-293cellsmeasuredwithRhod-4NWCal-ciumAssaykitandRhod-2AM.HEK-293cellswereseededovernightat40000cellsper100µlperwellina96-wellblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith100µloftheRhod-4NWcalciumassaykit,or5µMRhod-2AMat37°C,5%CO2incubatorfor1hour.Carbachol(25µl/well)wasaddedbyNOVOstar(BMGLabTech)toachievethefinal indicatedconcentrations.TheEC59ofrhod-4NWisabout0.8uM.

Description P/N : QtyRhod-4™NWCalciumAssayKit,MediumRemoval CQ6080 1 plate

CQ6081 10 platesCQ6082 100 plates

Rhod-4™NWCalciumAssayKit,1%FBSGrowthMedium CQ6090 1 plateCQ6091 10 platesCQ6092 100 plates

Rhod-4™AM CQ6061 5x50µgλexc./λem.:(Hydr.,Ca2+):530/555nm;Kd(Ca2+)=525nm CQ6062 10x50µg

CQ6063 20x50µgCQ6064 1 mg

■Rhod-4™AM-NoWash(NW)Thebrighestredfluorescentcalciumindicator

Rhod-2 is most commonly used among the redfluorescent calcium indicators. However, Rhod-2AMisonlymoderatelyfluorescentinlivecellsuponesterase hydrolysis, and has very small cellular calcium responses. Rhod-4™hasbeendevelopedto improve cell loading and calcium response while maintaining the spectral wavelength ofRhod-2.InCHOandHEKcellsRhod-4™AMhascellular calcium response that is 10 times more sensitivethanRhod-2AM.

Figure2.Rhod-4AMvsRhod-2AMinU2OS.U2OScellswereseededovernightat40000cellsper100µlperwell ina96wellsblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith100µlof5uMRhod-4AMorRhod-2AMinHHBSat37°C,5%CO2incubatorfor1hour.Thecellswerewashedwith2timeswith200µlHHBS,thenimagedunderfluorescentmicroscopeusingTritcchannel.


Calcium

Assays


Ratiometric calcium dyes

Description P/N : QtyFura-2AM FP-42776A 1 mg

FP-42776C 20x50µgFP-85312A 1ml(1mM)

Fura-PE3AM FP-AM603A 500µg

RepresentativeexperimentofangiotensinII(ANGII)-evokedchangesintheratiooffura2fluoresence(340/380nm) inadherentneonatal ratcardiomyocytes(NRC). Primary cultures of NRC loaded with Fura 2werestimulatedwithincreasingconcentrationsofANGII (10 pM-1 µM) either in the absence (control ; A) orpresenceofAA-861(10µM;B)orMK-571(100nM;C).■Indo-1AM

Indo-1AMhasashiftintheemissionfrom485nmto405nminthepresenceofcalcium.Indo-1AMcanbeusedwithasingleargon-ionlaserforexcitationandtomonitortwodifferentemissions.

Description P/N : QtyIndo-1AM FP-427755 500mg

FP-42775A 20x50µgFP-98180A 1ml(1mM)

Relatedproducts:4-bromoA-23187,Ca2+ionophoreforUVlight-exciteddyes FP-372221 1 mgA-23187(calcimycinorCalciumIonophoreIII)toequilibrateintracellularandextracellular[Ca2+] FP-28362B 5mg

Representativetracingsfromindo1-loadedmyocytesshowsimultaneousCa2+ transients (toppanels)andcelllength(bottompanels).Myocyteswerestimulatedat0.5Hzafter6hofnotreatment(control)(A),LPS(10ng/ml)(B),orLPSwithANGII(100nM)(C).

RelatedproductDescription P/N : QtyCalciumCalibrationkit FP-21527A 1kitPluronic®F-127 FP-37361A 1 gPluronic®F-127,20%solution(inDMSO) FP-69806A 1 ml

■Indo-PE3AMLeakageresistantformofIndo-1

Description P/N : QtyIndo-PE3AM FP-AM602A 500µg

■Fura-PE3AM&Fura-2AMLeakageresistantformofFura-2,apopularSetrometricCa2+ indicator

Fura-2AM may be useful inmicroplate studies, where cell lines with different properties arecomparedorwherescreeningtreatmentsleadtodifferencesinthenumberofcellsordyeloading.SomeofthelimitationsintheuseofFura-2appeartobeovercomebytheuseofglassbottommicroplates(Seepage78).

Reference:RobinsonJAetal.,Ratiometricandnon-ratiometricCa2+ indicators for the assessment of intracellular freeCa2+inabreastcancercelllineusingafluorescencemicroplatereader,JBiochemBiophysMethods.2004Mar31;58(3):227-37.

Fura-PE3 isan improvedversionofFura-2, that reducescell leakageand thus increasesdyeloading accuracy.

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Cell counting, viability & proliferation

Technical tipCellcountingisrequired» To monitor cells during cell cultures» For cell preparation or any cell experiment» To standardize cell samples for analysis. » Cellproliferation» Cytotoxicityassays

Severalmethods have been proposed, each fittingmore or less to each specificapplication:countingdeadcellsmaybeacceptableforthepreparationofcellextractsordesiredwhenonedonotwanttooperatewithhazardouscellsorforcytotoxicitystudy.Attheoppositedeadcellscountingisgenerallyprecludedforcellcultureandbioassays.Itmaybeusefultoquantitateonlyviablecells,oronlyfastproliferatingcells.

Interchimprovidesalargechoiceofcellassayscoveringstandardaswellasinnovativemethodsforgeneraltospecificcellassays.

SelectionguideProbe Principle Detection Method Dead Viable Proliferating Features/Advantages-DrawbacksTrypanblue Membraneexclusion Colorimetric ++ ++ ++ Cheap,buttimeconsuming,notscalable.

Microscopy Donotstateonviability.

Hoechst DNAprobeexclusion Fluorimetric ++ ++ +++ Cheap,Scalable,Nontoxic.Donotstateonviability.MorerapidthanMTT/XTT;unfixedorfixedsamples.

MTT Formazan dye, orange precipitate. Colorimetric - ++ +++

Popularmethod.Sensitive,Scalable.NontoxicIncreasedsolubilityandperformancefromMTTtoXTTandWST.

XTT SameasMTTbutmoresoluble. Colorimetric - ++ +++

WST Formazandye,soluble&not toxic - ++ +++

UptiBlue ratiometricblueprobe Colorimetric - +++ +++ Nosolubilizationstep(unlikeMTT).Applyalsotofor cell redox Fluorimetric adherentcells.SensitivitysimilartoMTT/XTT,but

easiertouseFluorimetry/SuperiorsensitivitytoMTT/XTT.

Calcein-AM Calceinaccumulationin Fluorimetric - +++ ++ Nosolubilizationstep(unlikeMTT/XTT).Adaptabletoacytoplasm widevarietyoftechniques,including:microplateassays,

invivocelltracing.Donotworkforbacteria.Mayaltersomecellfunctions.

GAPDH Release ofGAPDH coupledtoATPassay Bioluminescence - +++ + Measurement of Cell-Mediated (TCells,ADCC,NK) or

Complement-MediatedCytolsis.

CFSE Fluoresceinproteinlabeling Fluorimetric ++ ++ ++ Usefulwhenothermethoddonotworkproperly.Donotstateonviability.

AnnexinV AnnexinV/PhosphoSerine Fluorimetric + +++ + UsefulforApoptosisstudy.

LDH convertion in colored product - ++ + RecommendedforcytotoxicityassaysSerumInterference.

LuciferinSyst. ATPmeasure Luminescence - + +++ Pros:sensitivity/linearity.Cons:signaldependsoneachcellline,ontemperature

-3HThymidine DNAincorporationof Radioactivity - + +++ Cons:hazardous(radioelements).radioactivity

BRDU DNAincorporation Immunoassay - + +++51Cr release EU3+ Releaseofradioactivityby Radioactivity - - +++ Recommendedforcytotoxicityassays.

cytoplasm Cons:hazardous(radioelements).

Propidium 7-Membranepermeability Fluorimetric +++ - - Usedincombinaisonofgreenfluorescencedyelike.Iodide,AAD AnnexinV-FP488todiscriminatedeadcellsfromalivecells.

MicroPlatereaders&ImagingsystemsInterchimandBertholdcollaborationsupports furtheryourworks.Manyofourfluorescenceandluminescencereagentsandkitswerevalidatedwithinstruments.

*NightOWLLB983NC100

*MithrasLB940MultiModeReader


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Dual staining to detect live and dead cells

■Live/DeadMammalianViabilityAssayKitTwo-colorfluorescentstainingoflive(green)anddeadcells(red)

» DualDetection:Detectbothliveanddeadcellssimultaneously.» Simple&Fast:Requireonlya30-mindyeloadingtimeandthenmeasurewithoutwashing.» Economical:Performviabilityandcytotoxicityassaysatthesametime.» Versatile:Analyzewithflowcytometers,fluorescencemicroscopesorfluorescenceplatereaders.

TheViability/CytotoxicityAssayKitforLive/DeadCellsprovidesatwo-colorfluorescencestainingonbothliveanddeadcellsusingtwoprobesthatmeasuretworecognizedparametersofcellviability—intracellularesteraseactivityandplasmamembraneintegrity[Papadopoulos,1994].Thekitissuitableforusewithfluorescencemicroscopes,fluorescencemultiwellplatescannersandflowcytometersandotherfluorescencedetectionsystems.Theassayprinciplesaregeneralandapplicabletomosteukaryoticcelltypes,includingadherentcells[Vaughan,1995]andcertaintissues[Poole,1993],butnottobacteriaoryeast.Thisfluorescence-basedmethodofassessingcellviabilitycanbeusedinplaceoftrypanblueexclusion,51Cr releaseandsimilarmethods for determining cell viability andcytotoxicity. It is generally faster, lessexpensive, saferandamore sensitiveindicatorofcytotoxiceventsthanalternativemethods.EthD-IIIsharesthesamepropertywithEthD-IusedinLive/DeadViability/CytotoxicityAssayKit#486301andis40%brighteratintensitycomparedtoEthD-I.ValidityoftheLive/DeadViability/Cytotoxicityassayforanimalcellapplicationshasbeenestablishedbyseveralpublications.

Livecellsaredistinguishedby thepresenceofubiquitous intracellularesteraseactivity,determinedby theenzymaticconversionof thevirtuallynonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells,producing an intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). EthD-III enters cells with damagedmembranes andundergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em~495nm/~635nm).EthD-IIIisexcludedbytheintactplasmamembraneoflivecells.Thedeterminationofcellviabilitydependsonthesephysicalandbiochemicalpropertiesofcells.Cytotoxicevents thatdonotaffect thesecellpropertiesmaynotbeaccuratelyassessedusingthismethod.Backgroundfluorescencelevelsareinherentlylowwiththisassaytechniquebecausethedyesarevirtuallynonfluorescentbeforeinteractingwithcells.

Ifcellsarefirstfixed,andthenstained,theLive/DeadBacterialViability/Cytotoxicitykitcanalsobeconsidered.ToreplacethedyeCalceinAMthatwillonlystainthelivecells,theDMAO;aDNA-bindingdye,willstainbothintactanddamagedcellmembranes.

References:JImmunolMethods,177,101(1994).JCellSci,106,685(1993).JNeurosci,15,5389(1995).

Description P/N : QtyLive/DeadMammalianViabilityAssayKit FP-BF4710 1000 tests in microplate reader

Relatedproducts:DMAO,nucleistainforlivecells,2mMsolninDMSO FP-CA8150 1 mlEthidiumBromideIII,1mMsolution FP-BP9341 200µlMTT(λabs.(cleaved):650nm(550-600nm)) FP-65939A 1 gLive/DeadYeastViabilityAssayKit 486301 1kitbasedoncalcein-AMandPI. HelaCellsincubatedwithassaysolution.

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■Live/DeadBacterialViability/CytotoxicitykitTwocolorfluorescencestainingonbothlivebacteria(green)anddeadbacteria(red)

» DualDetection:Detectliveand deadbacteriacellsinacell population simultaneously. » Simple&Fast:15mindyeloading andmeasurewithoutwashing.» Economic:Performviabilityand cytotoxicity assays at the same time. » Versatile:Analysiscompatiable withflowcytometersand fluorescencemicroscopesusing popularsettingsforfluorescein and propidium iodide.

Viability/CytotoxicityAssayKit forBacteriaLive&DeadCellStainingKitprovides two-colorfluorescencestainingonboth live (green)anddead(red)bacteriausingtwoprobes,DMAOandEtD-III.DMAOisagreen-fluorescentnucleicaciddyethatstainsbothliveanddeadbacteriawithintactanddamagedcellmembranes.EtD-IIIisared-fluorescentnucleicaciddyethatstainsonlydeadbacteriawithdamagedcellmembranes.WithanappropriatemixtureofDMAOandEtD-III,bacteriawith intactcellmembranes isstainedfluorescentgreen,whereasbacteriawithdamagedcellmembranesisstainedfluorescentred.Thekitissuitableforusewithfluorescencemicroscopesandflowcytometers.Theassayprinciplesaregeneralandapplicabletomostbacteriatypes.

Acommoncriterionforbacterialviabilityistheabilityofabacteriumtoreproduceinsuitablenutrientmediathatisreferredtoasgrowthassays.Thiskityieldsresultsthatcorrelatewellwithgrowthassaysinliquidorsolidmedia.Undercertainconditions,however,bacteriahavingdamagedmembranesmaybeabletorecoverandreproduce—suchbacteriamaybescoredas"dead"inthisassay.Conversely,somebacteriawithintactmembranesmaybeunabletoreproduceinnutrientmedium,andyetthesemaybescoredas"alive".Therefore,thesesituationsneedtobeconsideredifavastdifferenceofliveanddeadbacteriacountsisobservedbetweenthisassayandgrowthassays.

Thiskitcanalsobeconsideredifcellslikemammaliancells,arefirstfixed,andthenstained.

Description P/N : QtyLive/DeadBacterialViability/Cytotoxicity FP-BU1040 1000 tests in microplate reader

Relatedproducts:Live/DeadYeastViabilityAssayKit 486301 1KitbasedonWST-8formazandye.Readat450nm(450-490nm)


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■Ethidiummonoazide,bromide(EMA)Selectively and covalently labels membrane-damaged or metabolicallycompromised cells in the presence of live cells

Ethidium monoazide bromide is a red fluorescent nucleic acid stain with aphotoaffinity label.The dye, after photolysis, binds covalently to nucleic acids.1 AfterphotocrosslinkingtoDNA,thewavelengths(λexc. /λem.=504/600nm)arecompatiblewithasimultaneousobservationofanothergreen indicator.Thedyehasbeenusedto"footprint"drugbindingsitesonDNA2tomodifyplasmidDNA,3,4 and to determine hemopoietic cell phenotype, function and position in the cell cycle.5Aparticularlyusefulapplicationofthedyeistoselectivelyandcovalentlylabeldeadcellsinthepresenceoflivecells.Sinceethidiummonoazidebromideis relatively impermeant to livecells, itselectively labelsDNA indeadcells inamixedpopulationof liveanddeadcells.Photolysisfollowingthedyeapplicationrenders thedeadcellDNAcovalently labeledwith thedye.Onecan thenwashandfixthecellpreparationandexamitbymicroscopyfluorescenceplatereaderor flowcytometry.Themajor advantageof thismethod is that researchers canavoid extensive manipulation of live pathogenic organisms.6At thedifferenceofpropidiumiodide,theethidiummonoazidebindscovalently,and,whenappliedtocellsbeforefixation,providesanindicationofwhatfractionoftheunfixedpopulationweremembrane-damagedormetabolicallycompromised.

References:1)J.Mol.Biol.92,319(1975)2)Euro.J.Biochem.182,437(1989)3)J.Biol.Chem.257,13205(1982)4)J.Biol.Chem.259,11090(1984)5)Cytometry11,610(1990)6)Cytometry,12,133(1991)7)PNAS,97,no.17,p.9504-9507(2000)

TwophotonfluorescentimageoflivePTK2cellsvitallystainedwithEMA:Alateprometaphasecellillustratingthehighselectivityofthestainforthechromosomes.

Description P/N : QtyEthidiummonoazide,bromide(EMA) FP-48256A 5mgλex./λem.(DNAbound):504/600nm;MW:420.3

■Propidiummonoazide(PMA)Selectivelyandcovalentlylabelsdeadcellsinthepresenceoflivecells

PMATMisaderivativeofEMA,butithassignificantlyhigherDNAbindingaffinityandiscellimpermeant.AsEMA,afterphotolysis,thedyeisconvertedtoafluorescentDNAstaincovalentlyboundtoDNA.

Description P/N : QtyPropidiummonoazide(PMA) FP-BZ9340 1 mgλex./λem.(DNAbound):510/610nm;MW:512

References:Nocker,A. et al.,Comparisonofpropidiummonoazidewithethidiummonoazidefordifferentiationoflivevs.deadbacteriabyselectiveremovalofDNAfromdeadcells.J.Microbio Meth. 67(2),310-320(2006).Nocker A. et al., UseofPropidiumMonoazideforLive/DeadDistinctioninMicrobialEcology,Applied and Environmental Microbiology,p.5111-5117,Vol.73,No.16(2007).PanY.,BreidtF., Enumeration of Listeria monocytogenesbyReal-TimePCRwithPropidiumMonoazideandEthidiumMonoazideinthePresenceofDeadCells,Appl. Environ. Microbiol.doi:10.1128/AEM.01198-07(2007).

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Live cell staining

Technical tipCalcein dye is a polyanionic derivate of fluorescein thatexhibits fluorescence that is essentially independentof pH between 6.5 and 12. The excitation and theemissionwavelengthsofcalceinare485nmand535nm,respectively.Itiswellretainedincells.Thesefeatureshavemade it a popular and versatile dye for various applications, including cell volume changes in neurons and other cells, endocytosis, gap junctional communication, membraneintegrityandpermeability,angiography,liposomes…It is worthy to notice that calcein is strongly quenchedby several ions, including Fe3+, Co2+, Cu2+ and Mn2+ at physiological pH (not by Ca2+ or Mg2+ ions). Ions levelsshouldthusbemonitored.

AM ester is membrane-permeant and enters readily cellmembranes. Intracellularesterasesconvert it intocalcein.The DMSO solution is more convenient (time saving,reduces solubilization variability) especially for morereproductiblescreeningassays. Fluorescence of calcein at pH9.0

■CalceinAMCellCounting&ViabilityAssayKitTheCalcein-AMKitprovidesasimple, rapidandaccuratemethod tomeasurecellviabilityand/orcytotoxicity.ThekitutilizescalceinAMfor thefluorometricdeterminationoflivingcellnumbers.Theamountofafluorescentdyereadat512nm,calcein,hydrolyzedbyesterasesincells,isdirectlyproportionaltothenumberofviablecellsinculturemedia.The96-wellmicroplateassayhasadetectionrangeoflessthan50cellstomorethan25000cellsperwell.Itcanbeusedfor384-wellplatesbyadding5µl(insteadof10µl)assaysolutionto50µlPBSsolutionperwell.Sinceesterasesandphenolredintheculturemediuminterferewiththefluorescencemeasurement,replacingthecellculturemediumwithPBSisnecessarypriortoaddingtheCalcein-AMassaysolution.Anincubationof10to30minutesgivessufficientfluorescenceintensityforthecellviabilitydetermination.

Features:» Suitableforproliferatingandnon-proliferatingcells» Idealforbothsuspensionandadherentcells» Non-radioactivemicroplate» Rapid(nosolubilizationstepasinanMTTassay)» Idealforhigh-throughputassays» Betterretentionandbrightnesscomparedtootherfluorescentcompounds(i.e.fluorescein)

Applications :» Celladhesion,chemotaxis,multidrugresistance,cellviability,apoptosis,cytotoxicity,...» Microplateassays,immunocytochemistry,flowcytometry,andinvivocelltracing

Description P/N : QtyCellCountingKit,calcein-AMbased 876981 500tests

876982 2x500tests

CalceinAM FP-895514 1 mgλex./λem.(cleaved):494/517nm;MW:994.9 FP-895515 20x50µg

CalceinAM,1mg/mlinanhydrousDMSO FP-855422 1 mlCalceinAM,4mg/mlinanhydrousDMSO FP-FI9820 100µlCalceinAM,5mMinanhydrousDMSO,PureGrade FP-JQ8140 200µl

Relatedproducts:AnnexinV-FluoProbes488,FlowCytometryGrade(495/519mm) FP-BH9390 100 testsPropidiumiodide,1mg/ml FP-36774A 10 ml


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Live cell staining

■CFDA,SEformicrobialandcellenumeration5-(and6-)-carboxyfluoresceindiacetate,succinimidylester(CFDA,SE)efficientlystainsgram-negativeandgram-positivebacterialgenerawithoutcausingundesirableeffectsoncelladhesionorviability.Thehighthroughputmethodusingmicroplatespectrofluorometryhasadetectionlimitofmid-105CFDA-stainedcells/ml.CFDA,SEtrackingtechniquehasapplicationsinbacterialtransport,publichealthmicrobiology,allowingthemovementofpathogentobemonitoredinterrestrial,aquatic,andevenfood-processingenvironments.Thetechniquemayalsobeusefulforstudyinginfectionandcolonizationbypathogensin vivo using animal models.

Reference:MarkF.etal. -DevelopmentofaVitalFluorescentStainingMethodforMonitoringBacterialTransport inSubsurfaceEnvironments,AppliedandEnvironmentalMicrobiology,October2000,p.4486-4496,Vol.66,No.10

Description P/N : QtyCFDA-SE(CFSE,GreenCellTrackingreagent) FP-52493A 25mgλex./em.(cleaved):495/519nm;MW:557

■UptiBlueCellViabilityAssayKitSubstrate:Resazurinλex./em.:540/590nmSensitive:100cells

Description P/N : QtyUptiBlueCellViabilityAssayKit UP669412 25ml

UP669413 100 ml

Applications

Cellproliferationassay

Detection of cell Growth of 4 Cell Lines usingUptiBlue

Kinetic/longtermassays

Kinetic reduction curvewithUptiBluewith platingdensityfrom500to10000cellsA549/ml.

Cytotoxicity assay

DeterminationofDoxorubicinLD50usingUptiBlueandXTT

Principle:theUptiBluedyeentersreadilyintocells,whereitelicitsawavelengthshiftofabsorbanceandastrongfluorescencerelatedtoredoxpotentialincell,informingoncellenergeticstate.

UptiBlueshowsexcellentcorrelationtoformazanandtritiatedthymidinetechniques,whilebeingmucheasierandsafertouse.ItespeciallyreplacesadvantagelyMTT/XTTinmanyapplications,fromcellcountingtoproliferationassayandcytotoxicitytesting.Furthermoreitallowslongerstudies.

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Live cell staining

■WST-8CellProliferationandCytotoxicityAssayKit» Colorimetricmicroplateassay» Ready-to-useone-bottlesolution» Safe : no radioisotope or organic solvent required» No toxicity to cells» Easyandfast:noharvesting,washingorsolubilizationsteprequired» MoresensitivethanMTT,XTT,MTSandWST-1

Reducedtoxicityofassaysolution:

CCK-8consistsofWST-8and1-methoxyPMSasanelectronmediator.Aftertheplateisincubatedfor1-4hoursintheincubator,theabsorbanceismeasuredin96or384-wellplate.Thewavelengthrangeforthemeasurementoftheabsorbanceisbetween450nmand490nm.Theamountoftheyellowcoloredformazandyegeneratedbydehydrogenasesincellsisdirectlyproportionaltothenumberofviablecellsinaculturemedium.ThesensitivityusingCCK-8ishigherthanthatusingMTTortheothertetrazoliumsaltsthatproducewater-solubleformazandyessuchasXTTorMTSforHeLacellsandHL60cells.Furthermore,thecellproliferationassaydatausingCCK-8correlateswiththatusingthe3H-thymidine incorporate assay.

Description P/N : QtyCKK-8CellCountingKit 899650 1000 tests

899651 3000 tests899654 10000 tests

Alsoavailable:MTTCellProliferationAssayKit 45547A 1000 testsMTTUltraPure FP-65939A 1 g

XTTCellProliferationAssayKit FX873A 1000 testsXTTUltraPure FP-409036A 1 g

WST-1CellProliferationAssayKit KS0790 96testsWST-1asstandaloneproduct F98883 100 mg

Cellproliferationassay:

Technical tipFormazanbasedCellViabilityAssayKit

MTTbasedassayisprobablythemostpopularcell viability assay. It has several drawbacksincluding toxicity, poor solubility that requiresan extraction step and limited sensitivity. Interchim provides those kits as well (seerelatedproducts)butrecommendsstronglytheWST-8assaykit,oralternatively theUptiBluereagent.


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Live cell staining

■aCella™-TOXBioluminescenceCytotoxicityAssay(GAPDH)MeasurementofCell-Mediated(TCells,ADCC,NK)orComplement-MediatedCytolysis

» Versatile :Assaycanberuninserumsupplementedmedia.» Homogenous-One-step,nowashassay.Assaycanberuninsameplateassamples.» FAST-Resultsin3-5minutes.» HighlySensitive-Candetectfewerthan500cells/well.» WorkswithPRIMARYCELLSfordeterminingcellCytotoxicity.» Non-destructiveassayallowsmonitoringofadditionalparameters.

aCella-TOXprovidesanewandhighlysensitiveassayusinga patented coupled luminescent technology for the detection of cytotoxicity(1). This assay quantitatively measures the release of Glyceraldehyde-3-Phosphate Dehydrogenase(GAPDH)fromPrimaryCells,mammaliancelllines,bacterialcells(1,2,3).aCella-TOXcanworkindifferentmediaformulationsand allows overnight assays while retaining its sensitivity.The sensitivity of aCella-TOX is also greatly enhancedby the coupled luminescent signal-amplification system(3-Phosphoglyceric Phosphokinase/ATP/Luciferase), whichyields a strong luminescent signal from even small amounts of released enzyme.

In the aCella-TOX reaction scheme the release ofGAPDHis coupled to the activity of the enzyme 3-Phosphoglyceric Phosphokinase (PGK) to produce ATP. ATP is detectedvia the luciferase, luciferin Bioluminescence methodology. Further,aCella-TOXisahomogeneouscytotoxicityassay;alternativelyindualmode,aCella-TOXcanmeasurecytotoxicityandcellviabilityinthesameplate.Culturesupernatantscanalsoberemovedfromtheoriginalplateandassayedinadifferentplate,allowingkineticsrunstobesetup.Theassayisnon-destructive,allowingthemonitoringofadditionalparameterssuchasgeneexpression.Themethodishighlygeneral,sinceallknowncellsexpresscopiousamountsofGAPDH,and,unlikeotherenzymes,GAPDHisveryreadilyreleasedfromthecytoplasmuponcelllysis.Usingspeciallyadaptedformulations,thesensitivityofthemethodcanbedrivenbelow1eukaryoticcell(2).

Applications :TheaCella-TOXmethodhasbeentestedwithmanymodesofcytolysis,including:» Cellularcytotoxicity(Tcells)» Complement(2,3), pore-forming agents» Antibiotic-mediatedlysisofbacteria» Detergentmediatedandmechanicallysis

References:1.Methodsandcompositionsforcoupledluminescentassays.UnitedStatesPatent6,811,990CoreyandKinders,issuedNovember2,2004.2.Corey,M.J.andKinders,R.J.(2005),DrugDiscoveryHandbook,Ed.ShayneCoxGad,pp.689-7313.Corey,M.J.,etalJournalofImmunologicalMethods207:43-51,1997.4.Corey,M.J.,etal.,JournalofBiologicalChemistry275:12917-12925,2000.5.OgbomoH.,etal.-BiochemicalandBiophysicalResearchComunications339(2006)pp375-379.6.Corey,J.andKinders,J.(2005),DrugDiscoveryHandbook,Ed.ShayneCoxGad,pp.689-731

Jurkatcellswereplatedatvariouscellconcentrationsperwell.NP-40cytotoxicagentwasaddedtoeachwell.TheaCella-TOXkitwasusedtodetectedG3PDHenzymerelease.DatapointsshowaverageRLUintriplicate.

Description P/N : QtyaCella-ToxbioluminescentCytotoxicityAssay CA4670 500testsKitContent:4xEnzymeAssayReagent,1xEnzymeAssayDiluent,Glyeraldehyde3-Phosphate(G3P),50xDetectionReagent,5.5xDetectionAssayDiluent,LyticAgent

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ATP, ADP, Phosphate & Pyrophosphate Assays

■ATPAssaykit,0.1to100pmolTodetectATPinbiologicalsamplesormonitoreATPdependentenzymeassays

Substrate:luciferinwithstabilizerλem. : 560nmSensitivity:0.1to100pmolATP - 30 min. signal

TheATPDeterminationKit, sensitiveassay,offersaconvenientbioluminescenseassay forquantitativedeterminationof smallamountsofATP.CatalysedbyfireflyluciferasethesubstrateD-luciferinisoxidizedinanATP-dependentprocessgeneratingchemiluminescenceat560nm(pH7.8):

luciferin+ATP+O2 Mg2+, luciferase Oxyluciferin+ATP+pyrophospate+CO2+light

The sensitive assay is optimized for fast determination of low levels of pre-existingATPorATP formed in kinetic systems.After a 10min incubation of the assay reagent,ATPconcentrationsdown to0.1pmolcanbeexactlydeterminedusing the linear luminescentsignaloftheluciferasereaction.Lossofluminescentsignalandsensitivityisobservedafterincubationtimesofmorethan30minutes.Ifyouareinterestedinatime-stableassay(i.e.forhighthroughputscreenings)withnearlyconstantluminescencesignalsoveraperiodofuptofourhours,useourSteadyGlowATPAssayKit.

Description P/N : QtyATPAssayKit,0.1to100pmolsensitive FP-S2841A 200-1000assays(10 ml)Eachkitcontains: FP-S2841B 600-3000assays(30 ml)ComponentA:FireflyLuciferase(readytouseglycerolstocksolution) FP-S2841C 2000-10000assays(100 ml)ComponentB:D-Luciferin(todissolveinreactionbuffer)ComponentC:DithiothreitolDTT(todissolveinreactionbuffer)ComponentD:ReactionBuffer(readytousebuffer)

Relatedproducts:ATPdisodiumsalt 00064A 25gReactionBufferasstandaloneproduct(ComponentDofATPAssayKit) CA3920 30 mlReactionBufferasstandaloneproduct(ComponentDofATPAssayKit) CA3921 100 mlARL-67156Ecto-ATPaseinhibitor CG2331 10 mg

Linear luminescence signal for ATP concentrationsdown to 0.1 pmol using theATP Determination Kit,sensitive assay.

■ATPAssayKit,SteadyGlowSubstrate:luciferinwithstabilizerλem. : 560nmSensitivity:10cells/well-10uMto0.1nMATP-4hsignal

Adenosine triphosphate (ATP) plays a fundamental role incellularenergenics,metabolicregulationandcellularsignaling.TheATPAssayKitprovidesa fast,simpleandhomogeneousluminescence assay for the determination of cell proliferation and cytotoxicity in mammalian cells. The assay can beperformedinaconvenient96-welland384-wellmicrotiter-plateformat. The high sensitivity of this assay permits the detection ofATPinmanybiologicalsystems,environmentalsamplesandfoods.ThisATPAssayKithasthestableluminescencesignalaslongas4hours.Ithasstableluminescencewithnomixingorseparationsrequired,andformulatedtohaveminimalhands-ontime.LinearluminescencesignalforATPconcentrationsfrom10µMto0.1nMwasdetectedupto5h(Z’factor=0.7)withoutsignaldecayed(abovefigshows20min,1,2,3,4,and5hrsignal).Theintegratedtimewas1sec.Description P/N : QtyATPAssayKit,SteadyGlow FN0630 96assays


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■ATPAssayKit,BrightGlowSubstrate:luciferinwithstabilizerλem. : 560nmSensitivity:10cells/well-3pmolATP2hincubationtime

Adenosine triphosphate (ATP) plays a fundamental rolein cellular energenics, metabolic regulation and cellularsignaling.TheATPAssayKit providesa fast, simpleandhomogeneous luminescence assay for the determination of cell proliferation and cytotoxicity in mammalian cells. The highsensitivityof thisassaypermits thedetectionofATPin many biological systems, environmental samples andfoods.ThisATPAssayKitcandetectaslowas10cells/well.It hasstable luminescencewithnomixingorseparationsrequired, and formulated to have minimal hands-on time.

ATPdoseresponseon96-wellwhiteplateusing2hincubationtime(Z’factor=0.6,Blue30min,red1h,andgreen2h).Theintegrationtimewas1sec.Thehalflifeismorethan1.5h.

Description P/N : QtyATPAssayKit,BrightGlow FN0640 96assays

■UniversalFluorimetricKinaseAssayKit,RedFluorescenceMostofcommercialproteinkinaseassaykitsareeitherbasedonmonitoringofphosphopeptideformationorATPdeletion.ForthekinaseassaykitsthatarebasedondetectionofphosphopeptidesonehastospendtimeandeffortstoidentifyanoptimizedpeptidesubstratewhiletheATPdepletionmethodsuffersvarious interferencesdue to theuseof luciferase thatare inhibitedoractivatedbyvariousbiologicalcompounds.TheUniversalKinaseAssayKitisbasedonthemonitoringofADPformation,whichisdirectlyproportionaltoenzymephoshphotransferaseactivityandismeasuredfluorimetricaly.Thiskitprovidesa fast,simple,andhomogeneousassay formeasurekinasesactivities.Thecharacteristicsof itshighsensitivity (<0.2uMADP),broadATPtolerance(1-300uM),non-antibodybased,non-radioactiveandno-washmethodtodetecttheamountofADPproducedasaresultofenzymeactivitymakeitanidealkitfordeterminingkinaseMichaelis-Mentenkineticsandforscreeningandidentifyingkinaseinhibitors.

» Universal:CanbeusedforanykinasesthatusedATPasphosphatedonor.» Continuous:Easilyadaptedtoautomationwithnomixingorseparationprotocols.» UseofNativesubstrates:Substratescanbeproteins,peptidesorsugars.» Non-Antibody-Based:Noantibodyisusedinthekit.

Description P/N : QtyUniversalFluorimetricKinaseAssayKit(540/590nm) CL9170 250assaysContains:ADPsensorbuffer,ADPSensor,ADPstandard,ADPAssayBuffer

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■ADPAssayKit,RedFluorescenceSubstrate:redfluorescentsubstrateλex./em. : 571/585nmSensitivity:0.2μMADPLargeRangeofATPTolerance:1-300μM.

ADPis involvedinmanybiologicalreactionssuchasproteinkinases.OurADPassaykitcanbeusedfor assaying protein kinase reactions universallyby monitoring ADP formation, which is directlyproportional to enzyme phosphotransferase activity and is measured fluorimetrically. This kitprovides a fast, simple and homogeneous assay for measuring ADP formation or depletion. Theassayiscontinuous,andcanbeeasilyadaptedtoautomation.Thekitisconvenient,requiringminimalhands-on time. Protein kinases are of interest toresearchers involved in drug discovery due to their broad relevance to diseases such as cancer andotherproliferativediseases,inflammatorydiseases,metabolic disorders and neurological diseases.Mostof commercialproteinkinaseassaykitsareeitherbasedonmonitoringofphosphopeptideformationorATPdeletion.OurADPAssayKitisbasedonthemonitoringofADPformation,whichisdirectlyproportionaltoenzymephoshphotransferaseactivityandismeasuredfluorimetricaly.Thiskitprovidesafast,simple,andhomogeneousassayformeasurekinasesactivities.

ADPdoseresponseon384-wellblackplatewith15,30minutesand1hourincubationtime(Z’factor=0.65).

Protein kinaseAdetectionwith incubation of the kinase in thepresenceofATPandkemptidepeptidesubstratefor30minutes.

Description P/N : QtyADPAssayKit,RedFluorescence CI4171 100 assays


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■PhosphateAssayKit,BlueFluorescenceSubstrate:proprietarysubstrateλex./em : 370/420nm

Phosphateisinvolvedinmanybiologicalreactions.Forexample,phosphatases,ATPasesandseveralotherenzymescatalyzereactionsinwhichinorganicphosphate(Pi)isreleasedfromasubstrate.ThisPhosphateAssayKithasbeendevelopedformeasuringtheactivityofanyPi-generatingenzyme.The kit is formulated to give the simplest detection of Pi, neither coupling enzymes nor hazardous radioactivemethods are involved.ThemeasurementofPi isbasedonourproprietaryfluorescentsensorthathasitsfluorescenceintensityproportionallydependentonphosphateconcentration.Unlikeotherphosphateassays,thiskitiseasytouse.Itisamixandreadformat,andcompatiblewithallthebiologicalbuffers.

Description P/N : QtyPhosphateAssayKit,BlueFluorescence JQ8120 1kit

■PhosphateAssayKit,RedFluorescenceSubstrate:proprietarysubstrateλex./em. : 540/590nmSensitivity:0.1μMphosphate

Cellsutilizeawidevarietyofphosphate(Pi)andpolyphosphate esters as enzyme substrates,second messengers, membrane structuralcomponents and vital energy reservoirs. Phosphate is involved in many biologicalprocesses. For example, phosphatases, ATPasesandseveralotherenzymescatalyzebiochemical reactions in which inorganicphosphate is released from a phosphoester substrate. Detection of many phosphoester–metabolizing enzymes is difficult becausesuitablesubstratesarenotavailable.Itusuallyhas been necessary to determine inorganicphosphate release using tedious colorimetric assays or radioisotope-based methods. ThisFluorimetric Phosphate Assay Kit has beendeveloped for measuring the activity of any Pi-generatingenzymeusingour redfluorescentphosphate sensor. The measurement of Pi isbasedon thechange in theabsorbanceandfluorescenceofournewphosphatesensor.Ourkitprovidesall theessential reagents includingphosphatesensor,phosphatestandardsandassaybuffer.Itcanbeusedtomeasurethekineticsofphosphatereleasefromphosphatases(suchasGTPasesandATPases)bycouplingthetwoenzymaticreactions.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomationwithnoseparationstepsrequired.

Phosphatedoseresponseon96-wellblackplatewith1hrincubationtime

Description P/N : QtyPhosphateAssayKit,RedFluorescence CI4161 100 assays

Alsoavailable:colorimetricphosphateassays*PhosphateAssay,MGmethod IS2790 1kit(600assays)Originalmolybdateandmalachytegreendyemethod.600-660nmreading.

PhosphateAssay,MGPlusmethod CI4211 1kit(1000assays)Improvedend-pointstablesignal(notpronetoprecipitation)

*The kit can also be used to estimate thephosphate content of proteins (phosphoserine or phosphothreoninepost-translationalmodifications,afteralkalinehydrolysis.

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■PyrophosphateAssayKit,BlueFluorescenceSubstrate:proprietarysubstrateλex./em. : 370/420nmSensitivity:0.3μM (30 pmoles) pyrophosphate

Pyrophosphate (PPi) are produced by a numberofbiochemicalreactions,suchasATPhydrolysis,DNA and RNA polymerizations, cyclic AMPformation by the enzyme adenylate cyclase andthe enzymatic activation of fatty acids to form their coenzyme A esters. The Pyrophosphate AssayKit provides the most robust spectrophotometricmethod for measuring pyrophosphate. This kituses our proprietary fluorogenic pyrophosphatesensor that has its fluorescence intensityproportionally dependent upon the concentration of pyrophosphate. Our assay is much easierand more robust than the enzyme-couplingpyrophosphatemethods that requireat least twoenzymes for their pyrophosphate detections. Thekitprovidesall theessentialcomponents forassaying pyrophosphate.

Description P/N : QtyPyrophosphateAssayKit,BlueFluorescence JQ8080 200assays


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NAD/NADH, NADP/NADPH Assay Kits

Nicotinamideadeninedinucleotide(NAD+)andnicotinamideadeninedinucleotidephosphate(NADP+)aretwoimportantcofactorsfoundincells.NADHisthereducedformofNAD+,andNAD+istheoxidizedformofNADH.ItformsNADPwiththeadditionofaphosphategrouptothe2'positionoftheadenylnucleotidethroughanesterlinkage.NADPisusedinanabolicbiologicalreactions,suchasfattyacidandnucleicacidsynthesis,whichrequireNADPHasareducingagent.Inchloroplasts,NADPisanoxidizingagentimportantinthepreliminaryreactionsofphotosynthesis.TheNADPHproducedbyphotosynthesis is thenusedas reducingpower for thebiosynthetic reactions in theCalvincycleofphotosynthesis.The traditionalNAD/NADHandNADP/NADPHassaysaredonebymonitoringofNADHorNADPHabsorptionat340nm.ThismethodsufferslowsensitivityandhighinterferencesincetheassayisdoneintheUVrangethatrequiresexpensivequartzmicroplate.ThesesNAD/NADH&NADP/NADPHAssayKitsprovideaconvenientmethod forsensitivedetection. Inaddition, thisassayhasvery lowbackgroundsince it is run in theredvisible rangethatsignificantlyreducestheinterferencefrombiologicalsamples.Theassayhasdemonstratedhighsensitivityandlowinterferencewith570nmexcitation590nmemission.

■NADHAssayKit,RedFluorescenceSensitivity:10nanomolesofNADHinsolution

The enzymes in the system specificallyrecognize NADH in an enzyme cyclingreactionwhichsignificantlyincreasesdetectionsensitivity.

NADPH dose response on 96-well black plate wasmeasuredwith1hourincubationtime(n=3)whilethereisnoresponsefromNADP(theinsertshowsthelowerdetection limit).

Description P/N : QtyFluorimetricNADPHAssayKit JQ7320 400 assays

■NAD/NADHAssayKit,RedfluorescenceSensitivity:100nM(10pmol/well)ofNADHin solutionλexc./em.: 570/590nm

The enzymes in the system specificallyrecognize NAD/NADH in an enzyme cyclingreaction.ThereisnoneedtopurifyNAD/NADHfrom sample mix. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.

Description P/N : QtyNAD/NADHAssayKit,Redfluorescence JQ7280 400 assays

NADH dose response on 96-well black plate wasmeasuredwith1hourincubationtime(n=3)whilethereisnoresponsefromNADPH.

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NAD/NADH, NADP/NADPH Assay Kits

■NADPHAssayKit,RedFluorescenceSensitivity:30nM(0,3nmol/well)ofNADPHinsolution

The enzymes in the system specificallyrecognize NADPH in an enzyme cyclingreaction. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.

NADPHdoseresponseon96-wellblackplatewasmeasuredwith 1 hour incubation time (n=3)whilethereisnoresponsefromNADP(theinsertshowsthelowerdetectionlimit).

Description P/N : QtyFluorimetricNADPHAssayKit,Redfluorescence JQ7330 400 assays

■NADP/NADPHAssayKit,RedfluorescenceSensitivity:10nM(1pmol/well)ofNADPHin solution

This NADP/NADPH Assay Kit provides aconvenient method for sensitive detection of NADP,NADPHandtheirratio.Theenzymesin the system specifically recognize NADP/NADPHinanenzymecyclingreaction.Thereis no need to purify NADP/NADPH fromsample mix. The enzyme cycling reaction significantlyincreasesdetectionsensitivity.

NADPH dose response on 96-well black plate wasmeasuredwithNADP/NADPHAssayKitwith 30minincubationtime(n=3)whilethereisnoresponsefromNADH.

Description P/N : QtyNADP/NADPHAssayKit,Redfluorescence JQ7300 400 assays


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Caspases study

■CaspasesFluorometricHTSAssayKits» HTS-compatible:Single-stephomogenousassayspecificallydesignedforHTS-baseddetection.» Fast :Fastenzymekinetics.» Sensitive:Theenzymaticreactionformsintenselygreenfluorescentrhodamine110(R110)product.ThelongwavelengthsofR110excitation andemissionminimizecellularautofluorescence(λex\λem=496/520nm).

Caspasesplay important roles inapoptosisandcell signaling.CaspasesFluorometricHTSAssayKitsare specifically designed forHTS-basedassays.Thekitsprovideahomogenousassaysystemforfastandhighlysensitivedetectionofspecificcaspaseactivitybyfluorescenceinenzymaticreaction or mammalian cells.

Theassaykitsincludeacaspaseinhibitorandcanbeusedasanegativecontrol.Also,R110dyeisprovidedinthekitforgeneratingastandardcurve,whichcanbeusedforquantifyingcaspaseactivity.

ThefluorogenicsubstrateR110-labeledcontainstwospecifictetrapeptidesandiscompletelyhydrolyzedbytheenzymeintwosuccessivesteps3, 4. Cleavageofthefirstpeptideresultsinthemonopeptideintermediate,whichhasabsorptionandemissionwavelengthssimilartothoseofR110,buthasonlyabout10%ofthefluorescenceofthelatter.HydrolysisofthesecondpeptidereleasesthedyeR110,leadingtoasubstantialfluorescenceincrease.

Thefluorogenicsubstrate(Ac-IETD)2-R110containstwoIETDtetrapeptidesandiscompletelyhydrolyzedbytheenzymeintwosuccessivesteps.Thefluorogenicsubstrate(Ac-LEHD)2-R110containstwoLEHDtetrapeptidesandiscompletelyhydrolyzedbytheenzymeintwosuccessivesteps.

Reference:1. Cell Death Diff.6,99(1999);2)J. Biol. Chem..274,11549(1999);3)J. Biol. Chem.275,288(2000);4)Biochemistry,38,13906(1999)

Description P/N : QtyCaspase-3FluorometricHTSAssayKit,DEVD-R110 FP-BR4930 1 ml

FP-BR4931 10 mlFP-BR4932 100 ml

KitComponents:Celllysis/assaybuffer,Enzymesubstrate(Ac-DEVD)2-R110,EnzymeinhibitorAc-DEVD-CHO,R110

Caspase-8FluorometricHTSAssayKit,IETD-R110 FP-BX1510 1 mlFP-BX1511 10 mlFP-BX1512 100 ml

KitComponents:Celllysis/assaybuffer,Enzymesubstrate(Ac-IETD)2-R110,EnzymeinhibitorAc-IETD-CHO,R110

Caspase-9FluorometricHTSAssayKit,LEHD-R110 FP-BX1530 1 mlFP-BX1531 10 mlFP-BX1532 100 ml

KitComponents:Celllysis/assaybuffer,Enzymesubstrate(Ac-LEHD)2-R110,EnzymeinhibitorAc-LEHD-CHO,R110

Relatedproducts:AnnexinV-FluoProbes488,FCMgrade(495/519mm) FP-BH9390 100 tests

Staurosporine(apoptosisinducer) 74146D 100µg74146E 1 mg

SeeBioScienceInnovationscatalog,Membraneapoptosisevents.

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Caspases study

■CaspasesFluorimetricandColorimetricAssayKitsContinuousmeasurementofthecaspaseactivity

» Fastenzymekinetics» Sensitive:Rhodamine110(496/520nm)minimizescellularautofluorescence» Versatile:Compatiblewithbothfluorometricandcolorimetricdetectionsystems.

TheprincipleisthesameasfortheCaspasesFluorometricHTSAssayKits.

Althoughfluorometricdetectionoftheendproductsispreferredbecauseofthesuperiorsensitivity,detectionbyabsorbanceisalsopossible.Infact,theextinctioncoefficientofR110is10timeshigherthanthatofp-nitroaniline(pNA),adyecommonlyusedinchromogenicsubstrates,makingR110-basedsubstratessignificantlymoresensitivethanpNA-basedsubstrates,evenbycolorimetricdetection.

Theassaykitincludesacaspaseinhibitorandcanbeusedasanegativecontrol.Also,R110isprovidedinthekitforgeneratingastandardcurve,whichcanbeusedforquantifyingcaspaseactivity.

Description P/N : QtyCaspase-3FluorometricandColorimetricAssayKit,z-DEVD-R110 FP-85785C 25tests

FP-85785B 100 testsKitComponents:Celllysisbuffer,Assaybuffer,Enzymesubstrate(Ac-DEVD)2-R110,EnzymeinhibitorAc-DEVD-CHO,R110

Caspase-8FluorometricandColorimetricAssayKit,IETD-R110 FP-BR4940 25testsFP-BR4941 100 tests

KitComponents:Celllysisbuffer,Assaybuffer,Enzymesubstrate(Ac-IETD)2-R110,EnzymeinhibitorAc-IETD-CHO,R110

Caspase-9FluorometricandColorimetricAssayKit,LEHD-R110 FP-BX1520 25testsFP-BX1521 100 tests

KitComponents:Celllysisbuffer,Assaybuffer,Enzymesubstrate(Ac-LEHD)2-R110,EnzymeinhibitorAc-LEHD-CHO,R110

Relatedproducts:Staurosporine,proteinkinaseinhibitor 74146D 100µgAnnexinV-FluoProbes488 FP-BH4140 500µl

SeeBioScienceInnovationscatalog,Membraneapoptosisevents


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Caspases study

■Caspase-6andGranzymeBbasedapoptosis/toxicityAssaysCytotoxicityismeasuredasfunctionsoffundamentalbiochemicalpathwaysleadingtocelldeath:-in the CyToxiLux®kit,cleavageofacellpermeablefluorogenicveryspecificsubstrateofcaspase-6,anestablishedinitialactivationstepinapotosis.Literature:NatureMed.8:185-189(2002)

-in the GranToxiLux™kit,cleavageofacellpermeablesubstrateforGranzymeB,involveinaearlyeventofcell-mediatedapotosis.GranzymeBthatisinactiveinlysosomalgranules,isactivatedinthepresenceofTargetcellsduring(degranulation),extremelyearly(beforePerforin).Literature:NatureMed.8:185-189(2002);MethodsMol.Biol.263:125-140(2004):J.Immunol.171:27-31(2003)

Theseassaysprovideanextremelyearlyquantitativeassessmentofcaspase-6andcell-mediatedcellularcytotoxicity.EspeciallyGrantoxiluxreplacesadvantageously the classic 51Crreleaseassayisanendstageofcellmediatedcytotoxicity,i.e.aftercelllysis.FurthermorethatcanbeusedbyFCMand microscopy yielding single cell measurements in complex populations.

Benefits:» Moreversatileinapplications:suitsCTL/NKandotherfactormediatedcytotoxicity,cytotoxicityinducedbyintracellularagentsor xenobioteics,physiologyandfateofeffectorcells» More rapid:co-incubationof0.3-2H(vs.4Hfor51Crreleaseassay)» Moresensitivethanthe51Crmethod:candetectrelativelyweakCTLresponsesagainstsubdominantepitopeswhereasthelattercannot.» Largestudyperiod:hourtodaysallowlongtermstudies,thatisusefulfornon-orslowproliferatingcells» compatiblewithmultiparametricFCM&Microscopyanalysisatthecelllevel,eveninmixedpopulations» Nosericinterferences:avoidthislimitationofLDHandFormazanmethods» Nopre-labelingofcells:avoidthislimitationof51Crmethod

Description P/N : QtyGrantoxilux cytotoxicity assay (Fluo.) BP8891 50testsMeasuretheGranzymeB(pathofcell-mediatedapoptosis)

Cytoxiluxcytotoxicityassay(Fluo.) BP8881 50testsMeasurethecaspase-6(classicpathofapoptosis)

Each kit contains sufficient reagents for 50 assays i n FCM. It may be applied also for microscopy with somemodifications(Caspase-6orGranzymeBSubstratesolution,Targetcellmarkerforusewithsinglelaserinstruments(Arion(488nm),Targetcellmarkerforusewithduallaserinstruments(Arion(488nm)andRed(633nm)),Resuspensionmedium,WashBufferbottle,Assay/Culturemedium.

Cytoxilux 51CrAssay

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DNA Damage & Condensation study

■UniversalChemiluminescentPARPAssayKit

» Chemiluminescent, non-radioactive format» Higherthroughput96testsize» Sensitivitydownto0.0025unitsofPARPperwell» Availableeitherwithahistonecoatedplateor histone reagent

Poly ADP-ribosylation of nuclear proteins is a post-translational event that occurs in response to DNAdamage. Poly (ADP-ribose) polymerase (PARP)catalyzes the NAD-dependent addition of poly (ADP-ribose) to adjacent nuclear proteins. PARP plays animportantroleinDNArepairbutcanalsoleadtoapoptosisbydepletingthecellularNADpool.Experimentalmodelshave shown that PARP inhibition prevents tissuedamage in animal models of myocardial and neuronal ischemia, diabetes, septic shock, and vascular stroke.Recent data implicate a synergistic function of Ku80andPARP-1inminimizingchromosomeaberrationsandcancerdevelopment.Universal96-wellPARPAssayKitmeasures the incorporation of biotinylated Poly (ADP-ribose)ontohistoneproteins ina96-well plate format.ThisassayisidealforthescreeningofPARPinhibitorsandformeasuringtheactivityofPARPincellandtissueextracts.

Applications :» IdentifyinhibitorsandactivatorsofPARPactivity» MeasurecaspaseinactivationofPARP» QuantitatelevelsofDNAdamageincellscausedbyavarietyofgenotoxicagents» MeasureactivityofPARPincellandtissueextracts

Description P/N : QtyUniversalChemiluminescentPARPAssayKitw/HistoneReagent HP9090 96testsUniversalChemiluminescentPARPAssayKitw/HistoneCoated HP9130 96tests

Relatedproduct:FITC-NAD FX8241 250µl


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■HTChemiluminescentPARP/ApoptosisAssayELISAassaykitformonitoringPARPactivitybeforeandduringapoptosis

Sensitivitydownto0.1mUnitsofPARP-lessthan500cells/well

Duringapoptosis,PARP-1whichcatalyzes theNADdependentadditionofpoly(ADP-ribose)(PAR)ontovariouscytoplasmicandnuclearproteins,is cleaved from about 116 kDa to 85 kDa. HT PARP/ApoptosisAssayis ideal for measuring the activity of PARP in cell extracts before andduring apoptosis. The HT PARP/Apoptosis Assay is an ELISA whichsemi-quantitatively detects PAR deposited onto immobilized histoneproteinsina96-wellformat.Ananti-PARmonoclonalantibody,goatanti-mouse IgG-HRPconjugate,andHRPsubstrateareused togenerateachemiluminescentsignal.Thus,absorbancecorrelateswithPARPactivity.EtoposideisatopoisomeraseIIinhibitorthatstabilizesthisenzymeafteritcleavesDNA.Itisincludedasacontrolapoptosisinducer.

Description P/N : QtyHTChemiluminescentPARP/ApoptosisAssay CP1990 96testsKitcontent:PARPBuffer,activatedDNA,PeroxyGlow™A&B,Histonecoatedstripwells,PARPHSA,NAD,Antibodydiluent,anti-PARmonoclonalantibody,HRPconjugatediluent,Etoposide.

GraphicalrepresentationofanexampleChemiluminescentreadoutofaPARPstandardcurve.

■HTChemiluminescentPARGAssayKit» Chemiluminescent format» Higherthroughput96testsize» Sensitivitydownto50pgofPARGperwell

Poly(ADP-ribose) glycohydrolase (PARG) degrades poly(ADP-ribose) (PAR)polymerssynthesizedbypoly(ADP-ribose)polymerase(PARP1).WhenactivatedbyDNAstrandbreaks,PARP1usesNADasasubstratetoformADP-ribosepolymerson itself andonspecificacceptorproteins suchashistones,DNApolymerases,DNAligases,p53,andFos.ThesepolymersareinturnrapidlydegradedbyPARG,a ubiquitously expressed exo- and endoglycohydrolase. Excessive activation ofPARP1leadstoNADdepletionandcelldeathduringischemiaandotherconditionsthat generate extensive DNA damage. PARGmay maintain the active state ofPARP1 by continuously removing inhibitory ADP-ribose residues from PARP1.The regulation of PARG activity may therefore, influence PARP-mediated celldeath.First,PARGinhibitioncouldslowtheturnoverofPARandtherebylimitNADdepletion.Second,PARGinhibitioncouldpreventtheremovalofPARfromPARP1.BecausePARP1isinhibitedbyextensivepoly-(ADP-ribosyl)ation,PARGinhibitorscouldtherebyindirectlyinhibitPARP1activity.PriorworkhasshownthatthePARGinhibitorgallotannincanmarkedlyreducedeathofastrocytesafteroxidativestress.HTChemiluminescentPARGAssayKitmeasuresthelossofbiotinylatedPARfromhistonesattachedtostripwellsina96wellformatandisidealforthescreeningofPARGinhibitorsandformeasuringtheactivityofPARGincellextracts.

Applications :» IdentifyinhibitorsandactivatorsofPARGactivity» MeasureactivityofPARGincellandtissueextracts

Description P/N : QtyHTChemiluminescentPARGAssayKit JZ4060 96tests

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■FlowTACS™ApoptosisDetectionKitsIdentifyandquantitateapoptoticcellsinculture

DNAfragmentation isacommittedstep inapoptosis,andthe labelingof3’endsprovidesaneasymeasureofcellsundergoingapoptosis.CellsmayalsobeanalyzedforDNAcontentusingtheincludedpropidiumiodide/RNaseAsolution.TheFlowTACS™KitalsoprovidesTACS-Nuclease™to generate positive controls for calibration. The FlowTACS™Kit uses fixed cells, allowing you to safelyworkwith cells that are infectedwithbiohazardousagents.Also,samplesmaybestoredconvenientlyduringtime-courseexperiments.

Thiscompletekitprovidesall the reagents required for labeling including twopermeabilization reagents, labelingandstopbuffers, labelinganddetectionreagents,andTACS-Nucleaseforgeneratingpositivecontrolswithyourownsamples.

Features:» Fast.Requireslessthan3hourstocomplete.» Exclusive, non-toxicTACSSafeTdT™buffer-sodiumcacodylatefree.» Uniquebuffersystemproducesmoreconsistentlabeling.» Worksonfixedcells.» IncludesexclusiveCytonin™permeabilizationreagent.» IncludesTACS-Nucleasesolutionforpreparingsample-dependentpositivecontrols.

Description P/N : QtyFlowTACS™ApoptosisDetectionKits 512510 60samplesKitcontent:Permeabilizationreagents,Optimizedcation,TACS-Nuclease,Labelingbuffer,Detectionreagents,Propidiumiodide/RNasesolution,Nucleotidemix,Fluorescentlabel,Labelingenzyme,Counterstain

■Hoechst33342Cell-permeantbis-benzimidethatbindstoDNAwithfluorescenceenhancement(λexc./λem.:350/461nm).

Reference:DebbaschC,etal.,Mitochondrialactivityandglutathioneinjuryinapoptosisinducedbyunpreservedandpreservedbeta-blockersonChangconjunctivalcells. Invest Ophthalmol Vis Sci.2001Oct;42(11):2525-33.

Description P/N : QtyHoechst33342,10mg/mlinwater FP-59046A 10 ml


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Mitochondrial events

■JC-10MitochondriaMembranePotentialAssayKit» IncreasedSignalIntensity:Largerassaywindow» Increasedsolubility:MuchbetterwatersolubilitythanJC-1» ConvenientandRobust:Formulatedtohaveminimalhands-ontime» Versatileapplications:Compatiblewithmanycelllinesandtargets

JC-10andJC-1ComparisononEffectofCampotothecininducedmitochondriamembranepotentialchangeinJurKatcells.Jurkatcellsweretreatedwith10µM

camptothecinfor4hours.JC-1andJC-10dyeloadingsolutionwasthenaddedtothewellsfor30minutes.ThefluorescentintensityforbothJ-aggregatesandmonomeric

formsofJC-1andJC-10wasmeasuredatEx485nm/Em520and595nm.

Although JC-1 is widely used inmany labs, its poorwatersolubilitymakes it hard touse for someapplications.Evenat1µMconcentration,JC-1tendstoprecipitateinaqueousbuffer.JC-10hasbeendevelopedtobeasuperioralternativetoJC-1wherehighdyeconcentrationisdesired.Comparedto JC-1, our JC-10hasmuchbetterwater solubility. JC-10is capable of entering selectively into mitochondria, andchanges reversibly its color from green to orange asmembrane potentials increase. This property is due to thereversible formation of JC-10 aggregates upon membranepolarization that causesshifts inemitted light from520nm(i.e., emission of JC-10 monomeric form) to 570 nm (i.e.,emission of J-aggregate). When excited at 490 nm, thecolor of JC-10 changes reversibly from green to greenishorange as the mitochondrial membrane becomes morepolarized. This JC-10 Mitochondrial Membrane PotentialAssay Kit enable you to monitor mitochondrial membranepotentialchangesusingasimplemicroplatereaderwhileallthe other commercial JC-1 assay kits require the use of aflowcytometer.Ourkitprovides themost robustmethod tomonitormitochondrialmembranepotentialchanges,andcanbereadilyusedforscreeningalargecompoundlibrary.

Description P/N : QtyJC-10MitochondriaMembranePotentialAssayKit DT2420 5plates(96-or384-well)JC-10 CL0440 5mg

Relatedproducts:CCCP 091640 500mgJC-1,asstandaloneproduct FP-52314A 5mg

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Mitochondrial events

■SafranineODynamic and quantitative approach of changes ofmitochondrialmembranepotential(DeltaPsi(m))ΔΨm:

» Suitablefordynamicstudies of energization» Allowsdirect assessmentofbothsubstrate- dependent, electron transport-mediated ΔΨmandATP hydrolysis-supported ΔΨm.» AvoidlimitationofJC-1uptakewhentheplasma membranepotentialdecreases

Fluorescenceisfollowedat485nmexcitationand586nmemission.Unlike the behavior of JC-1, safranine O was rapidly and completelyreleasedwhenmitochondrialwerede-energizedwithFCCP.ThedecreasedfluorescenceofsafranineOresultingfromquenchingofsafranineOafteruptakebyenergizedmitochondriameansthatlowfluorescencecorrespondstohighΔΨm(Feldkamp,2004).

Description P/N : QtySafranineO N12820 25g

N12821 100 gReference:FeldkampT. et al.–Assessmentofmitochondrialmembranepotentialinproximaltubulesafterhypoxia/reoxygenation,AM.J.Physiol.RenalPhysiol.,288:F1092-F1102(2005)

Relatedproducts:Ouabain 050538 1 g

■GlutathioneDetectionKit,MonochlorobimaneGSHdepletionfollowupduringearlystageofmitochondrion-associatedapoptosis

» Simple:Compatiblewithastandardorfluorescenceplatereader.» Fast:Around1hourassaytime.

Diminishedcellularglutathione(GSH)leveloccursattheearlystageofmitochondrion-associatedapoptosispathwayduetoGSHefflux.GSHdepletionfurtherleadstocytochromecreleaseandcaspase3induction(1,2)MCBGlutathioneDetectionKitutilizesathiol-reactivedyemonochlorobimane(MCB),whichisessentiallymonfluorescentuntilitreactswithathioltoformabluefluorescentproduct(λabs/λem=380/461nm).ByincubatingcellularlysatewithMCB,theintensityofthefluorescentsignalgeneratedfromtheassayreflectstheamountofGSHpresentinthecells.

Reference:1)FASEBJ.12(6),479(1998);2)Biochem.Soc.Trans.28,56(2000).

Description P/N : QtyGlutathioneDetectionKit,Monochlorobimane FP-BU1410 100 testsKitcontent:Celllysisbuffer,Monochlorobimane(MCB),GSTpositivecontrol

Relatedproduct:Monochlorobimane,asstandaloneproduct FP-38980A 25mg

■LiveCellGlutathioneTransferaseActivityKitλabs/λem:380/461nmSamplesize:200cells

Thisnewkitprovidesreagentsandmethodstosimplyandquicklymeasureglutathionetransferaseactivityinlivecells,tissuesorcelllysatesamples.ItallowsintracellularglutathioneS-transferasedetectionbysimplyaddingafluorogenicreagentmCBtothecellculturemediumorlysatetoformGSH-mCBcomplexes.Unlikeotherbimanessuchasmonobromobimane,monochlorobimaneappearstoformanadductexclusivelywithGSH.ThisprocedurehasbeenusedtomeasureGSHcontentofculturedneuralcellsandintissuehomogenatesand,indeed,severallaboratorieshaveusedthisapproachtomeasuretheGSHcontentofthecytosolicfractionofliverorinintacttissues.IthasbeenfoundthatmonochlorobimanereadilyenterscellstoformafluorescentGSHmono-chlorobimaneadductthatcanbemeasuredfluorometricallyandthatthisreactioniscatalyzedbyglutathioneS-transferase.Description P/N : QtyLiveCellGlutathioneTransferaseActivityKit BQ2350 100 assaysKitcontains:Monochlorobimane,CellLysisBuffer,L-Glutathione,GlutathioneS-transferase


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Reactive Oxygen Species (ROS)

Oxidativemetabolismstudy(ROS,NO)

TheproductionoffreeradicalsprimarilyresultsfromO2catchedbycellsandreducedinmitochondria.98%isfullyutilizedbycytochromecoxidasetoformwater,butthisenzymecanreleasepartlyreducedspecies.Otherrespiratorychainenzymes,andinparticularcomplexesIandIII,alsoproducepartlyreducedoxygenspeciesincludingsuperoxide.Thesereactiveoxygenspeciescanreactwithnitricoxidetoproducereactivenitrogenspeciesincludingperoxynitrite.Asignificantproportionofthereactiveoxygenandnitrogenspeciesdiffusewithcontrolledrateintothecytosol,wheretheyreactwithvariousmolecules,lipids,proteins,sugarsandnucleotides.Butamajorportionremainsinthemitochondrionwheretheycausesoxidativedamage.Whentheelectrontransferefficiencydecreases,moreradicalsareproduced,andsomorecytosolicproteinsaredamaged.Moreover,oxidativeandnitrativedamageofmitochondrialproteinsaddstoOXPHOSdysfunctionfurtherexacerbatingfreeradicalproduction.AprotectivemechanismagainstROSisSODmetabolism.

Enhancedoxidativestressoccursinnumberdegenerativediseases.Inhuman,ROSareconsideredtobeoneofthemaincausesofaging-relateddiseases,Parkinsonsdisease,Alzheimersandothervascular-damage-relatedbraindiseases,Cancer,Artherioschlerosisanddiabetes.In plants, theSODactivity is increased by the use of herbicides such as paraquat, by theSO2 concentration in the atmosphere, by drought, or by exposure to high concentration of zinc andmagnesium.ROSprobeshavehighselectivityandsensitivityinenzymaticoxidationreactions,favorisingtheirusefordiagnosticanalysis.Also,peroxidaseisacommonenzymeforsignalamplificationinimmunoassays(EIA).

InterchimprovidesseveralROSprobesforfluorimetryandcolorimetry,chromogenic,fluorogenicorluminogenic.

Technical tip

SelectionguideFluorescentROSprobes

ReactiveOxygenSpecies(ROS)P/N : Probes HydrogenPeroxide Hydroxy radical Hypochlorousacid Peroxyl radical Peroxynitrite anion Superoxideanion

H202 H0- HOCl COO- ONOO- O2-FP-83775 Dihydrorhodamine123 + + +FP-46731 H2DCFDA + + + +FP-46915 Lucigenin* +FP-97233 Coelenterazine + +FP-38544 MCLA* + +Dihydrocalcein AM + (1O2) + +Dihydroethidium - +24200A tMPVCA7170 HPF + +CA7270 APF - + + - + -U3238A ADHP +

*ROS-generatingEnzymeDetectionkitsMyeloPeroxidasedetectionkit HemoproteinofPMNscells/Cl-oxidationtoHOClCatalasedetectionkit antioxidantenzyme/decomposesH2O2Cis-Parinaricacid fattyacidtomonitorlipidperoxidationMalachiteGreenIT produceaHydroxylradicalburstuponirradiationSuperOxideDismutase(SOD) convertsO2-● into H2O2andO2

*Otheroxidativespecies:aldehydesSSAOdetectionkit deamination/formaldehyde,methylglyoxalMonoAmineOxidase(MAOA&B) oxidateavarietyofneurotransmitters/aldehydes

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■H2DCFDA,Carboxy-H2DCFDA,H2DCFDA-SEH2DCFDA is widely used to detect reactive oxygen species (hydrogen peroxide, ONOO- ) in cells (neutrophils, macrophages). Colorless (λexc./λem.:258/none;EC:11000)andnonfluorescentuntiltheacetategroupsarehydrolyzedbyintracellularesterasesandoxydationoccurswithinthecell,giving thehighlygreenfluorescent2',7'-dichlorofluorescein (DCF#FP46629;λabs.λem.:495/529nm).Applications includeROSdetection,viabilityandcytotoxicityassays,apoptosis.ItcanbeusedwithPropidiumiodidetofollowoxidantproductionandnuclearinjury.Carboxy-H2DCFDAisanindicatorforROS.Onpenetrationintothecells,carboxy-H2DCFDAisdeacetylatedbyintracellularesterases,resultinginanenhancedcellularretentionoftheprobe.Thefluorescenceoftheoxidizedformofcarboxy-H2DCFDAismeasuredwithafluorescenceexcitationof485nmandemissionat535nm.H2DCFDA,SEisanaminereactiveformoftheH2DCFDAtoprepareoxidation-sensitiveconjugates,likedextran.Thisallowsdetectionofoxidativeburstinphagocyticcells.

Reference:JiyoungK.andRaghubirS.,Calcium-MediatedActivationofc-JunNH2-TerminalKinase(JNK)andApoptosis inResponsetoCadmiuminMurine,Macrophage, Toxicological Sciences200481(2):518-527

Description P/N : Qty2',7'-Dichlorodihydrofluoresceindiacetate(H2DCFDA) FP-467312 100 mgCarboxy-2',7'-Dichlorodihydrofluoresceindiacetate(CH2DCFD) FP-46634A 25mg2',7'-Dichlorodihydrofluoresceindiacetate,succinimidylester(H2DCFDA-SE) FP-59031A 5mgλexc./λem.(hydr.&oxid.)(pH4):495/529nm;EC:38000M-1cm-1

λexc./λem.(hydr.&oxid.)(pH8):504/529nm;EC:107000M-1cm-1

■Coelenterazine(native)Coelenterazine is a sensitive chemiluminescentmarker for detecting both superoxide and peroxynitrite. It has no significant effect on xanthineoxidase–dependentoxygenconsumption,endothelialcellhydrogenperoxiderelease,orendothelium-dependentrelaxation.Coelenterazineemitschemiluminescence(Em=466nm)onoxidationbysuperoxide.

Reference:Margaret T. et al.- Chemiluminescent Detection of Oxidants in Vascular Tissue, Lucigenin But Not Coelenterazine Enhances Superoxide Formation,Circulation Research. 84:1203-1211(1999)

Description P/N : QtyCoelenterazine(native) UP972333 1 mg

■MCLASuperoxideorsingletoxygenchemiluminescentprobe

MCLAlikecoelenterazineisasuperioralternativetolucigeninforsuperoxidedetection.Lucigenincanreportedlysensitizesuperoxideproduction,leadingtofalse-positiveresults.AnadditionaladvantageofMCLAisthatitspHoptimumforluminescencegenerationisclosertothephysiologicalnear-neutralrangethanarethepHoptimaofluminolandlucigenin.MCLAgenerateschemiluminescence(Em=455nm)uponreactionwithsuperoxide.

Reference:.TeranishiK,et al.Enhancedchemiluminescenceof6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-onebyattachmentofacyclomaltooligosaccharide(cyclodextrin).Attachmentofcyclomaltononaose(delta-cyclodextrin)."Carbohydr Res338,987-93(2003).KondoM.et al.,Theabilityofneonatalandmaternalerythrocytestoproducereactiveoxygenspeciesinresponsetooxidativestress.",Early Hum Dev66,81-8(2002).SakuraiT.et al.,SuperoxideproductionintheisletofLangerhansdetectedbytheMCLAchemiluminescencemethod."Methods Mol Biol196,203-9(2002)

Description P/N : QtyMCLA FP-38544A 5mg2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one,hydrochloride


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■Dihydroethidium(hydroethidine)MeasurementofO2

–

Thesuperoxideoxidizesdihydroethidiumtoaspecificfluorescentproduct(oxyethidium)thatdiffersfromethidiumbythepresenceofanadditionaloxygen atom in its molecular structure. Exposure of dihydroethidium to hydrogen peroxide or peroxynitrite caused no formation of oxyethidium from dihydroethidium.Fluorescencedetectionat590nm(emission)and530nm(excitation)isusedtomonitoroxyethidiumproduction.

Reference:DebbaschC,etal.,Mitochondrialactivityandglutathioneinjuryinapoptosisinducedbyunpreservedandpreservedbeta-blockersonChangconjunctivalcells.Invest Ophthalmol Vis Sci. 2001Oct;42(11):2525-33.

Description P/N : QtyDihydroethidium,specialair-freepackaging FP-52492A 25mg

FP-52492B 10x1mgDihydroethidium,5mMinDMSOFP-R5919A 1mlDihydroethidiumFP-524929 20x50µg

■Dihydrorhodamine-1,2,3(DHR123)Measurementofperoxonitrite(ONOO–)

ThelevelofONOO–canbemeasuredbymonitoringtheoxidation ofdihydrorhodamine-1,2,3(DHR123),usingmicroplatereaderatexcitationandemissionwavelengths of485nmand530nm,respectively.Itisnonfluorescentuntiloxidizedtothemitochondrialproberhodamine123.

Reference:JiYoungLee,et al.,Inductionofendothelialapoptosisby4-hydroxyhexenal,Eur. J. Biochem.271,1339-1347(2004)

Description P/N : QtyDiHydroRhodamine123,5mMstabilizedsolutioninDMSO FP-R6805A 1 mlDihydrorhodamine123 FP-83775A 10 mgDihydrorhodamine123,air-freepackaging FP-IT397A 10 x 1 mgDihydrorhodamine123,5mMinDMSO FP-R6805A 1 ml

■DihydrocalceinAThiscellpermeantprobeisoxidizedtothecalceinwithbettercellretentionthanH2DCFDA.

Reference:KellerA,et al.Analysisofdichlorodihydrofluoresceinanddihydrocalceinasprobesfor thedetectionof intracellular reactiveoxygenspecies.Free Radic Res. 38:1257–1267(2004)

Description P/N : QtyDihydrocalceinAM FP-BA1970 1 mg

FP-T7996A 20x50µg

■trans-1-(2'-Methoxyvinyl)pyrene(tMPV)Sensitivesingletoxygenchemiluminescenceprobe

trans-1-(2'-Methoxyvinyl)pyrene is themostsensitivesingletoxygenprobe thatcouldbeused todetectpicomolequantitiesofsingletoxygen inchemicalandbiologicalsystems.Furthermore,thishighlyselectiveprobedoesnotreactwithotheractivatedoxygenspeciessuchashydroxylradical,superoxideorhydrogenperoxide.Itgenerateschemiluminescence(Em=465nmin0.1MSDS)uponreactionwith1O2.

Reference:NatBiotechnol24,95-9(2006).PlantandCellPhysiology46(6):947-954(2005)MethodsEnzymol133,569-584(1986)

Description P/N : Qtytrans-1-(2'-Methoxyvinyl)pyrene(353/401nm) FP-24200A 1 mg

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■Hydroxyphenylfluorescein(HPF)AssayKitFluorescent Hydroxyl (•OH)/Peroxynitrite(ONOO-) assay

» QuenchedCellpermeabledye» Canbeusedwithcelllysates,tissuehomogenates» Onestep,nowash,homogenousassay» Canmonitormultipletimepointstofollowrealtimekinetics» Non-destructivecellbasedassayallowsmonitoringofadditionalparameters

A novel probe, Hydroxyphenyl fluorescein (HPF)is a highly selective probe for the detection ofhighlyReactiveOxygenSpecies(hROS). It isacellpermeable highly sensitive fluorescent probe forhydroxyl radical (OH•), and peroxynitrite (ONOO-) detection. Ithas little reactivity towardsotherhROSsuch as : hypochlorite (-OCl), singlet oxygen (021), superoxide (02

-•), hydrogen peroxide (H202), nitric oxide(NO•),andalkylperoxide(RO2•).

Reference:Ken-ichiSetsukinai,et al.: Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. J. of Bilogical Chemistry,Vol.278,No.5,IssueofJanuary31,pp.3170–3175,2003

Description P/N : QtyHydroxyl/PeroxynitriteDetectionKit(HPF) CA7170 150tests

Relatedproduct:FeTPPS,specificperoxynitritescavenger 888810 250mg

■APF(forhROSdetection)SelectiveIndicatorforHighlyReactiveOxygenSpecies

» Canmonitormultipletimepointstofollowreal time kinetics» Quenchedcellpermeabledye» One-step,nowash assay» AdaptableforHighThroughput format» Non-destructivecellbasedassayallowsmonitoringofadditionalparameters» Applications-FluorescencePlateReader/FluorescentMicroscope/FlowCytometry

Anewnovelprobe,Aminophenylfluorescein(APF)developedbyTetsuoNaganoet.al.(1), is a general selective indicator for the detection of highly reactiveoxygenspecies(hROS).TheprobehaslittlereactivitytowardsotherROSsuchas:singletoxygen(O2

1),superoxide(O2-•), hydrogen peroxide

(H2O2),nitricoxide(NO•),andalkylperoxide(RO2•)(seetablebelow)1.APFisacellpermeableindicatorthatcanbeusedtodetectHydroxylRadical(-OH),Peroxynitrite:(ONOO-) and hypochlorite (-OCl)productionincells.

ROS(RFU) APF(RFU) DCFH-DA(RFU)Ex:499Em:515 Ex:500Em:520

HydroxylRadical:•OH 1200 7400Peroxynitrite:ONOO- 560 6600Hypochlorite : -OCl 3600 86OxygenRadical:1O2 9 26Superoxide:O2

-• 6 67Hydrogen Peroxide : H202 <1 190NitricOxide:NO <1 150AlkylperoxylRadical:ROO. 2 710Autoxidation <1 2000Reference:1-SetsukinaiK.,et al.,DevelopmentofNovelFluorescenceProbesThatCanReliablyDetectReactiveOxygenSpeciesandDistinguishSpecificSpecies.ThejournalofBiologicalChemistryVol.278,No.5,IssueofJanuary31,pp.3170–3175,2003

Description P/N : QtyAminophenylFluorescein(APF) CA7270 150tests


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■HypochloriteDetectionKitFluorescentOCl(Hypochlorite)assay

» QuenchedCellpermeabledye» CanbeusedwithCellLysates,TissueHomogenates» OneStep,NowashHomogenousassay» Canmonitormultipletimepointstofollowrealtimekinetics» Non-destructivecellbasedassayallowsmonitoringofadditionalparameters

Thetwonovelprobes,Aminophenylfluorescein(APF)andHydroxyphenylfluorescein(HPF)areselectiveforthedetectionofhighlyreactiveoxygenspecies(hROS).TheyoffergreaterselectivityandstabilitythandoesH2DCFDA.BothprobeshavelittlereactivitytowardsotherROSsuchas:singletoxygen (02

1),superoxide(O2-•), hydrogen peroxide (H202),nitricoxide(NO•),andalkylperoxide(RO2•).HPF/APFarecellpermeableandcanbeused

incombinationtodetecthypochlorite(-OCl)productionincells(seefig1).Hypochloritecanbedetectedbyloadingtwosamples,onewithAPFandtheotherwithHPF.HypochloriteproductionisvisualizedbyincreaseinfluorescenceofAPFloadedcellsandnoincreaseinfluorescenceinHPFloaded cells.

ReactivityProfileofAPF/HPF:ROS(RFU) HPF(RFU) APF(RFU) DCFH-DA(RFU)

λex./em.:499/515nm λex./em.:499/515nm λex./em.:500/520nmHydroxylRadical:•OH 730 1200 7400Peroxynitrite:ONOO- 120 560 6600Hypochlorite: -Ocl 6 3600 86OxygenRadical:1O2 5 9 26Superoxide:O2

-• 8 6 67Hydrogen Peroxide : H202 2 1 190NitricOxide:NO 6 1 150AlkylperoxylRadical:ROO 17 2 710Autoxidation 1 1 2000

Description P/N : QtyHypochloriteDetectionKit(HPF,APF) CA7250 150tests


H2O2doseresponseon384-wellblackplatewith30minutesincubationtime(n=3).TheinsertshowsthelowlevelsofH2O2detection.

■ADHPHydrogenperoxideAssayKit"Readandmix"sensitiveH202DetectionKit

Sensitive:10picomoles of H2O2 in solution.Hydrogen peroxide (H2O2)isareactiveoxygenmetabolicby-productthat serves as a key regulator for a number of oxidative stress-related states. It is involved in anumberof biological events thathavebeenlinkedtoasthma,atherosclerosis,diabeticvasculopathy,osteoporosis,anumberofneurodegenerativediseasesandDown'ssyndrome. Perhaps the most intriguing aspect of H202 biologyis the recent report that antibodies have the capacity to convertmolecularoxygenintohydrogenperoxidetocontributetothenormalrecognition and destruction processes of the immune system. Measurement of this reactive species will help to determine howoxidativestressmodulatesvariedintracellularpathways.HydrogenPeroxideAssayKitusesourRedperoxidasesubstratetoquantifyhydrogenperoxide insolutions, incellextractsand in livecells. Itcanalsobeused todetect a varietyof oxidaseactivities throughenzyme-coupledreactions.Thekit isanoptimized"mixandread"assaythatiscompatiblewithHTSliquidhandlinginstruments.Description P/N : QtyADHPHydrogenperoxideAssayKit CL2580 500assays

Alsoavailable:ADHPHydrogenperoxide/PeroxidaseAssayKitdualmode,candetectH2O2or peroxidase activity. U3238A 500tests10-acetyl-3,7-dihydroxyphenoxazine(ADPH)λabs/λem.:563/587nm FP-39423B 25mg

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■MyeloperoxidaseDetectionKit» Readout:Fluorescenceorabsorbance» Canmonitormultipletimepointstofollowkinetics» One-step,nowashassay» AdaptableforHighThroughputformat» Sensitive

Myeloperoxidase (MPO) is a highly cationic glycosolated hemoprotein that has amolecular weight of 144kD. The hemoprotein consists of two dimers linked via adisulfidebridge.Eachdimmeriscomposedofaheavy(53kD)andlight(15kD)subunit.Each heavy chain contains an independently acting protoporphyrin group containing a central iron. MPO is present in the azurophilic granules of polymorphonuclearleukocytes(PMNs)andisuniquetoneutrophilsandmonocytes.However,monocytescontainonlyonethirdoftheMPOfoundinPMN’s.MPOutilizesH202producedbytheneutrophils tooxidizeavarityofaromaticcompoundstogivesubstrateradicals forbactericidalactivity.Thisenzymeisuniquehoweverinthatitcanoxidizechlorideionstoproduceastrongnonradicaloxidant,HOCl.HOClisthemostpowerfullbactericidalproducedbyneutrophils.Excessiveproductionoftheseradicalscancauseoxidativestressleadingtooxidativetissueinjury.

H2O2+Detectionreagent(non-fluorescent)+MPOfluorescentanalog. Ex./Em.:530-571/590-600nm

MPOstandardcurvewasseriallydilutedin1XReactionBuffer.Reactioncocktail(RC)waspreparedasdescribed(withoutEPOinhibitor).Next 50 μL ofMPO standard and 50 μL ofRCwasaddedtoindividualwellofa96wellblackplates.Theplatewasincubatedat roomand temperature in thedark.DatacollectedEx.:530nmEm.:590nm.

References:JNeuralTransmSuppl23:55–72.(1987)Proc.Nat.Acad.Sci.U.S.A.,85,4934–4938(1988)Biochem. Pharmaco., 17,1285–1297.ll(1968)J. Biochem., 79,1297–1299(1976)Biochem. Pharmaco., 27,1995–2000(1978)

Description P/N : QtyMyeloperoxidaseDetectionKit CF2980 500tests

■CatalaseDetectionKitCatalase is an antioxidant enzyme that catalyses the decomposition ofhydrogen peroxide (H202) to water and oxygen. Catalase is ubiquitouslyexpressedinmammalianandnon-mammalianaerobiccellscontainingthecytochromesystem.Theenzymehasbeen isolated fromvarioussources,includingbacteriaandplantcells (1-3).Catalaseactivityvariesgreatly fromtissuetotissue.Highestactivityisseeninliverandkidney,whilelowestactivityis seen in connective tissue (3).Ineukaroticcells,catalaseinconcentratedinorganelles called peroxisomes (4). Theproductionofhydrogenperoxide ineukaryoticcells isanendproductresultofvariousoxidasesandsuperoxidedismutasereactions.Accumulationof H202canresultincellulardamagethroughoxidationofproteins,DNAandlipids thus resulting in cell death and mutagenisis (8-11). H202 role in oxidative stressrelateddiseaseshavebeenwidelystudied(8,12). TheCatalasedetectionkitissensitiveassaythatutilizesanon-fluorescentsubstrate, 10-Acetyl-3, 7-dihydroxyphenoxazine (ADHP, 530/590 nm), todetect H202substrateleftoverfromthecatalasereaction(5-6). CatalaseactivitydetectedusingtheCatalasekit.

Thereactioncontained20µMH202(final)perwellandtheindicatedamountsofcatalasein1XreactionBuffer.Thereactionwasincubatedfor30minutesatroomtemperature.Next100µLofReactioncocktailwasaddedtoeachwellandthereactionincubatedforanother10minutesinthedarkatroomtemperature.

Description P/N : QtyCatalaseDetectionKit CA7280 500tests

References:1.Physiol.Rev.,50,319-375(1970).2.AnalyticalBiochemistryVol.245,Issue1,1February1997,Pages55-60.3.Physiol.Rev.,50,319-375(1970).4.ProgressinBiophys.Mol.Biol.,72,19-66(1999).5.AnalBiochem253,162(1997).6.J.ImmunolMethods202,133(1997).7.ArchivesofBiochemistryandBiophysics,431:138-144(2004).8.J.Biol.Chem.,Sep1999;274:26217-262249.FEBSLett.,442,65-69(1999).10.FEBSLett.,414,552-556(1997).11.FEBSLett.,473,177-182(2000).12.CancerRes.,61,2766-2733(2001).


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■Cis-ParinaricAcid(CPA)Measurementoflipidperoxidation

Cis-parinaricacid(CPA)isafluorescentpoly-unsaturatedfattyacidusedasaprobetodirectlymonitorlipidperoxidation.Fluorescencemeasurementisusing318nmexcitationand420nmemissionfilters.

Description P/N : QtyCis-ParinaricAcid FP-46900A 10 mg

■SODAssayKitwithcolorimetricsubstrateWST-1» Measures100%inhibitionbySOD » pH independentIC50 determination » Convenient96-wellmicroplatecolorimetricassay » Low-backgroundnoise measurement

Superoxide dismutase (SOD), which catalyzes thedismutationofthesuperoxideanion(O2

-•) into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. In order to determinethe SOD activity, several direct and indirect methodshavebeendeveloped.Amongthesemethods,anindirectmethod using nitroblue tetrazolium (NBT) is commonlyusedduetoitsconvenienceandeaseofuse.However,there are several disadvantages to the NBT method,such as poorwater solubility of the formazan dye andtheinteractionwiththereducedformofxanthineoxidase. SODAssayKit-WSTallowsveryconvenientSODassayingbyutilizinghighlywater-solubletetrazoliumsalt,WST-1thatproducesawater-solubleformazandyeuponreductionwithasuperoxideanion.TherateofthereductionwithO2•-arelinearlyrelatedtothexanthineoxidase(XO)activity,andisinhibitedbySOD,asshownintheFigureabove.Therefore,theIC50(50%inhibitionactivityofSODorSOD-likematerials)canbedeterminedbymeasuring the decrease in the color development at 440 nm.

Description P/N : QtySODAssayKit S07411 100 tests

S07410 500tests

Relatedproducts:WST-1asstandaloneproduct F98883 100 mgNADPH Q91330 40-wells

■MalachitegreenisothiocyanateLocalizedproductionofhydroxylradicalsbyamine-reactiveprobe

Thisnon-fluorescentphotosensitizerprobe(λexc./em.628nm/none)canbeconjugatedtospecificantibodies.Enzymesandotherproteinswithin~10Åofthebindingsiteofthemalachitegreen–labeledantibodycanthenbeselectivelydestroyedbyproductionofhydroxylradicalsuponirradiationwithlong-wavelengthlight.

References:StresserDM.etal.,DrugMetabDispos30,845-52(2002)TolosaL.etal.,Lifetime-basedsensingofglucoseusingenergytransferwithalonglifetimedonor.".AnalBiochem250,102-108(1997)BeermannA.etal.,"Chromophore-assistedlaserinactivationofcellularproteins."MethodsCellBiol44,715-732(1994)

Description P/N : QtyMalachitegreenisothiocyanate FP-98782A 10 mg

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Aldehyde oxidation study

■MonoamineOxidaseA&BDetectionKitMonoamineoxidase(MAO) isaflavin-containingenzymethatcatalysestheoxidationofavarietyofamine-containingneurotransmitterssuchasserotonin,norepinephrine,epinephrineanddopaminetoyieldthecorrespondingaldehydes.MAOexistsintwoisoforms,namelyMAO-AandMAO-B,whicharetheproductsoftwodistinctgenes.MAO-AandBexhibit different specificities to substratesand inhibitor selectivities.Extensive studieshavebeenpreformed to characterize theirproperties.MAO-Aactspreferentiallyonserotoninandnorepinephrine,andisinhibitedbyclorgyline.MAO-Bactspreferentiallyon2-phenylethylamineandbenzylamineandisinhibitedbydeprenylandpargyline.Localizedintheoutermitochondrialmembrane,theseenzymesarefoundthroughoutthebody.Oftenonlyoneformoftheenzymeispresentinaspecificorganand/orwithinaspecificcelltype.Inadditiontotheirroleinregulatingneurotransmitters,theseenzymesarealsoinvolvedinprocessingbiogenicaminesincludingtyramine.

Substrate+O2+H2O+MAO-A/B aldehyde+NH3+N2O2

H2O2+Detectionreagent(non-fluorescent)+Peroxidase Resorufin(fluorescent) Ex./Em.:530-570/590-600nm

Description P/N : QtyMonoamineOxidaseA&BDetectionKit CA7290 500tests

■SSAODetectionKitFluorescentSemicarbazide-SensitiveAmineOxidaseDetectionKit

Semicarbazide-sensitiveamineoxidase(SSAO)isacommonnameforawidelydistributedenzymeinnature.Inmanthisenzymeispresentinthevascular system and circulates in plasma.SSAO’sfunctionalrolehasbeensuggestedtobeinvolvedin:apoptosis,atherogenesis,celladhesion,leucocytetrafficking,glucosetransportandlocalproductionofhydrogenperoxide.ReportsofelevatedlevelsofSSAOhavebeenreportedincongestiveheartfailure,diabetesmellitus,alzheimer’sdiseaseandvariousotherinflammatorydiseases.

Furthermoreby-productsofSSAOdeamination,suchasformaldehydeandmethylglyoxal,havebeenproposedtobeinvolvedinpathogenesisofcancer, aging and atherosclerosis.

Benzylamine+O2+H2O+SSAO Benzaldehyde+NH3+N2O2

H2O2+Detectionreagent(non-fluorescent)+Peroxidase Resorufin(fluorescent)

Description P/N : QtySSAODetectionKit CA7310 500tests


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Nitric Oxide (NO) assay

■DAF-2diacetate» Cellpermeable» Nowash homogenous assay» Real time detectionofNOSactivity» NOandNO2

– detection limit : ~5nM

NOscavengingcanbemeasuredbymonitoring4,5-diaminofluorescein (DAF-2).DAF-2,asaspecificNOindicator,selectively trapsNObetweentwoaminogroupsinitsmolecule,andyields triazolofluorescein,whichemitsgreenfluorescencewhenexcited at490–515nm.Thediacetateformiscell-permeablederivativeofDAF-2.DAF-2diacetatecanbeusedtodetectNOSactivityincellcultureortissuesections.ThisreagentisnotspeciesspecificandcanalsobeusedtodetectNOSactivityinplantcells(butnotinbarleyaleuronecells1).References:1)JournalofExperimentalBotany200657(3):463-470

Description P/N : QtyDAF-2diacetate S03720 100µgNOSDetectionkit CA7150 125µg(2.22mg/ml)

CA7151 250µg

Relatedproducts:SNAP,photoactivableNOdonor FP-71646A 25mgSpermineNONOate,pHcontrolledNOdonor FP-M16259 10 mgCarboxy-PTIOpotassiumsalt,specificNOscavenger 199500 5mgL-NMMA,NOsynthaseinhibitor FP-85524A 50mg

■DAF-FMDAF-FMisimportantreagentforquantitatinglowconcentrationsofnitricoxideinsolution.ThiscompoundisessentiallynonfluorescentuntilitreactswithNOtoformafluorescentbenzotrizole(495/515nm).ThediacetateformismembranepermeantandisdeacetylatedbyintracellularesterasestoDAF-FM.

» Fluorescence independentofpHabovepH5.5» SignificantlymorephotostablethanthatofDAF-2» NOandNO2

– detection limit : ~3nM versus

Description P/N : QtyDAF-FM FP-R1227A 1 mgDAF-FMdiacetate FP-R1228A 1 mg

■2,3-Diaminonaphthalene2,3-Diaminonaphthalenereactswithnitrosonium,whichisformedfromNO,toformthefluorescentdye1H-naphthotriazole(365/415nm).1,2Usingthismethod,10nMto10µMofnitrite(NO2-)canbedetectedandthedetectioniscompatiblewith96-wellformat.3

References:Luminescence14,283(1999)MethodsEnzymol.268,105(1996)Anal.Biochem.214,11(1993)

Description P/N : Qty2,3-Diaminonaphthalene FP-04832F 100 mg

■NBDMethylhydrazinefornitriteassayNBDmethylhydrazine(N-methyl-4-hydrazino-7-nitrobenzofurazan)reactswithNO2

–inthepresenceofmineralacidsleadstoformationoffluorescentproducts(468/537nm).NBDmethylhydrazinehasbeenusedtoquantitatenitriteinwaters.

Reference:AnalChem71,3003-3007(1999)

Description P/N : QtyNBDMethylhydrazine FP-R1315A 50mg

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Fluorescent DNA Assay

■EvaGreen-DNAquantificationinsolution» Linearresponseontherangeof1–100ng» Samplesize:50µl» Standardfilterset:λexc./λem. : 490/520nm» RequiressmallamountsofDNAanddye

Theintensityoffluorescence(relativefluorescenceunits)isproportionaltothetotalamountofDNApertuberatherthantheconcentrationofDNAinthetube.

WangW.etal.hasusedtheEvaGreenforDNAquantificationon25µlsamplewithlinearresponsefrom1ngto100ng(AnalyticalBiochemistry,Volume356,Issue2,Pages303-305(2006)):FigureAandB.AstronglinearrelationshipisobservedwhentheamountofDNAislessthan100ng(Fig.A),andthisisveryreproducible(Fig.B).

RelationshipsbetweenfluorescenceintensityandtheamountofDNApertube:Triplicatesamplesof_DNAintherangeof1–1000ng(A)or0–100ng,(B)wereaddedtoPCRtubescontaining1.25µlofEvaGreen(20Xconcentrate),andwaterwasaddedtomakeafinalvolumeof25µl.

Description P/N : QtyEvaGreen20XinPBS BI1790 5x1ml(1000assays)EvaGreen20000XinDMSO CA6770 1ml(200000assays)

Relatedproducts:DNAfromlambdaphage,asstandardforDNAquantification UP947860 100µgHoechst33258,forhighconcentrationDNAquantification FP-61248A 100 mgDNAdosagebyUVwithonly5µlsample,usingIMAplates seedescriptionpage72

OtherapplicationoftheEvaGreenintheBioScienceInnovationcatalog.


Biochem

istry Assays


Protein assays

Sensitivity: 0.5µg/ml(MicroBCAssay)20µg/ml(BCAassay)

1-25µg/ml(maxsens.protocol)50-1500µg/ml(Broadrangeprotocol) 50-160pg/mlpeptide,proteins

Linearrange: 1-250µg/ml(MicroBCAssay)20-2000µg/ml(BCAassay)

1-25µg/ml(maxsens.protocol)50-1500µg/ml(Broadrangeprotocol) 100ng/ml-160µg/ml

Reading 562nm 570-610nm 405-500/560-610nm

Comments : advantages

ThemostreliablemethodCompatiblewithmostdetergents,bases,DNA,lipidsBroader linearityLowestvariationsbetweenproteins

Quick (1-10 min)Compatiblewithreducers

UltimatesensitivityandlinearityPeptide dosageBiodegradableRobust,amenabletoN-termsequencing,MSandfunctionnalassays

Comments : limitations Need37°Cincubationorlongertime Limitedcompatibility(reducers, acids, some detergents) Limitedcompatibility

Description P/N : QtyBCAssayproteindeterminationkit UP4080A 1kit-1LBicinchoninicacidbasedmethod-590nm. UP4080B 1kit-250mlContains:1Lreagentsufficientfor500/5000tests(tube/µplate),and10x1mlBSAstandard2mg/ml

MicroBCAssayproteindeterminationkit UP75860A 1kit-1LVersionofBCAssaywithsensitivity0.5µg/ml. UP75860B 1kit-50mlContains:1Lreagentsufficientfor500/3400tests,and10x1mlBSAstandard2mg/ml

CooAssayproteindeterminationkit UPF86400 1kit-1LModifiedBradford(Coomassiebasedmethod). UPF86401 1kit-250mlContains:1Lreagentsufficientfor500/4000tests(tube/µplate),and10x1mlBSAstandard2mg/ml

LavaPeppeptide&proteinsassay CH4191 1KitEpicoccononebasedmethod. (upto2000tests)Contains:Dyeconcentrate10X,Bufferconcentrate10X

Interchimproposes3methodstoassayproteinsinmicroplates:

Colorimetric/BCA method :BCAssay&MicroBCAssay

Colorimetric/Bradfordmethod:CooAssays()

Fluorescent/Epicocconone:LavaPepmethod

Description P/N : QtyGlucose assay BD1850 1Kit(120ml)Hexokinase/G6PDHbasedmethod.Readingat500nm.Linearto400mg/dL.Forserum,plasmaorurine.5minprocedure.

Creatinineassay BP9991 1Kit(30ml)Enzymaticmethod.Readingat546nm.Linearto30mg/dL.Forserum,plasmaandurinesamples.

β-Hydroxybutyrateassay AL2230 1Kit(60ml)Enzymticmethod.Readingat505nm.Linearto4.5nmol/L.Forserumorplasma.

Glucose assay U67120 1Kit(40tests)Hexokinase/G6PDHbasedmethod.Readingat340nm.Linear1-80µg/ml.Forfoodstuffandbewerage.

Glycerol assay R51065A 1Kit(4x10tests)Enzymaticmethod.Readingat330/334/365nm.Onecomponent.Forfoodstuffandbewerage.

Cholesterolassay R5756A 1Kit(4x10tests)EnzymaticCHOD/PAPmethod.Readingat546nm.Forfoodstuffandbewerage.

Contactusforotheranalytesinbiologicalsamples(Calcium,Bilirubin,HDL,Urea,.../Maltose,Fructose,Starch,Malate,Lactate,...).

Interchimprovidesawholerangeofbiochemistryassaysforclinicalchemistry(Blood,Urine,CerobroSpinalfluids,...)aswellasforothersamples(food,soil,water...)analysedinagro-alimentaryindustryorenvironementstudy.Hereisashortselectionofassaykits.

Biochemistry tests for biologicals samples

Ordertogetherthefollowingreagenttorendertheseproteinassayscompatiblewithanyinterferingsubstance!

ProteinPreparationkitRéf.:R5594A500ml

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Immuno Assays

ImmunoAssays have been popularized using ELISA technique (Enzyme Linked ImmunosorbentAssay), especially using peroxidase (HRP) oralkaline phosphatase (AP) with colorimetric substrates. Interchim offering include not only highly sensitive colorimetric substrates, but alsoluminescentsubstrates,thathavespreadthankstheirincomparablesensitivity,andfluorescentimmunoreagents that can increase the dynamic range,sensitivityandnotablyallowformultiplexedanalysis.Wefinallyprovidebiotin/streptavidinreagents,thatarerecommendedtogetanamplificationofthesignal.Additionnally,biotinlabelisgeneralymoreflexibleandquickertocalibrateusingfewlabeledstreptavidinsformanydifferentassays

The luminol was introduced as a convenientand effective chemiluminescent substrate,overcoming the performances (and first, thesensitivity) of classical insoluble chromogenicsubstrates (incl. OPD, TMB, DAB). Theprincipleconsistsof thegenerationof lightbytheby-products of the chemical reaction fromperoxidase upon the substrate. The emissionof glow is then recorded by luminometer at 425 nm (in tubes, or in wells of ELISAmicroplates).Theuseofluminescentsubstratesis most recommended for quantitative assays requiring extended dynamic range of detection or qualitative assays requiring the bestachievabledetectionlimit(highestsensitivity).

Technical tip

Interchim provides a new chemiluminescent substrate for ELISA techniques, that improvessignificantlythesensitivityofdetection,aswellthestabilityandreproducibility-somecrucialpointsin screening experiments.Not only signal is at equal or higher to your usual reagent, but thebackgroundislower.Asaresult,UptiLightELISAsubstrateachievesunsurpassedSignaltoNoise(S/N)ratios.

SensitivitycomparisonwithUptiLightandcompetitorsSignal tonoise inELISAwithUptiLightUSELISAHRP (#996201)comparedwithcompetitorsPEF,BEF,AEA,andPBP.ELISAwasperformedwithcoatedMouseIgGdetectedbyantiMouseIgG(H+L)-HRP(#UP446330),thentheECLLuminescentsubstrates,preparedaccordingtheirrespectivesupplier.LuminescencewasrecordedwithMithras(BertholdTechnolgies)with0,1secintegrationtime,aftera5minpreincubationperiod.DatasareplottedasSignaltoNoiseratios(S/N) for each testedHRPAbconcentration.Reducedbackgroundandhigher sensitivitywasfoundwithUptiLightreagent.

Photostability of light emission with UptiLight USLuminescencewas recorded with 0,1 sec integration time after a 1, 5, 10 or 30minpreincubationperiod.

Description P/N : QtyUptiLightELISAHRPUltraSensitivityChemiluminescentSubstrate. 996201 60ml

996202 120ml996203 300 ml

Alsoavailable:UptiLightELISAHRPHighSensitivityChemiluminescentSubstrate. 36349A 200mlAcosteffectivereagentforroutineanalysis,whenonlypicomoledetectionisrequired.

ELISA - Luminescent detection (ECL)

■UptiLightELISAHRPChemiluminescentSubstrateIncrease10timesthenetsignalofyourusualluminolreagent,forultra-sensitiveHRPbasedimmunoassays.

» Highsignalwithminimalbackground» Sensitivityinthepicoandfemtorange» Signalstableupto1hour» GreatforHTSapplications» Reagentstable>18monthsat4°C» Easytouse:mix,incubate5min,read

SeealsochemiluminescentkitsforAssay#JQ6760page 18.


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ELISA - Luminescent detection (ECL)

Relatedproducts:

SelectedHRPlabeledsecondaryantibodies(antiIgs)Host a Specificity Biotinlabeled Qty HRPlabeled Qty

*AntiHumanantibodiesGtIgG Anti-HumanIgG,minXBov,Hrs,Mssrprot. UP892650 0.5mg UP783493 1 mgGtIgG Anti-HumanIgG,Fcfragmentspecific,minXBov,Hrs,Mssrprot. 722640 1 mg 802020 1 mlMsMab Anti-HumanKappaLightChain UPB91830 0.5mg UPB91850 0.5mgRtMab Anti-HumanLambdaLightChain UPB91870 0.5mg UPB91890 0.5mgRtMab Anti-HumanIgGGammaChain UPB92190 0.5mg UPB92210 0.5mgRtMab Anti-HumanIgMMuChain UPB91910 0.5mg UPB91930 0.5mgRtMab Anti-HumanIgDDeltaChain UPB91990 0.5mg UPB92010 0.5mgRtMab Anti-HumanIgEEpsilonChain UPB92030 0.5mg UPB92050 0.5mgRtMab Anti-HumanIgAAlpha1&2Chain UPB92150 0.5mg UPB92170 0.5mg

*AntiMouseantibodiesDkIgG Anti-MouseIgG(H+L),minXBov,Ck,Gt,GP,SyHms,Hrs,Hu&Rb,Shpsrprot. 946220 0.5ml UP973171 0.5mgGtIgG Anti-MouseIgG(H+L),minXHu,Bov,Hrs,Rb,Swsrprot. 668610 1.5ml UP215731 1 mgRbIgG Anti-MouseIgG(H+L),minXHusrprot. 794090 1 ml 794560 1 ml

AntiRabbitantibodiesDkIgG Anti-RabbitIgG(H+L),minXBov,Ck,Gt,GP,SyHms,Hrs,Hu&Ms,Rt,Shpsrprot. UP944471 0.5mg 338370 0.5mlGtIgG Anti-RabbitIgG(H+L),minXHusrprot. 812230 1.5ml UP687714 1 mgGtIgG Anti-RabbitIgG(H+L),minXMs,Rtsrprot. UP668621 1 mg UP669631 1 mg

*AntiRatantibodiesGtIgG Anti-RatIgG(H+L) 740470 2ml UP399892 1 mgSecondaryantibodiesareavailablewithdifferentpreadsorbtions,formats(Fab'2),raisedin6hosts,andagainst22targetIgspecies.Pleaseinquire.

HRP(strep)avidinreagentsDescription P/N : QtyHRPlabeledStreptavidin UP395888 1 mgHRPlabeledAvidin 35445A 2mgHRPlabeledNeutralizedAvidin UP36570A 1 mg

■LuminometricAlkalinePhosphataseAssayKitAlkalinePhosphataseAssayKitusesaproprietary luminogenicphosphatasesubstrate, toquantifyalkalinephosphataseactivity immobilizedonsurfaces,i.e.ELISA,aswellasinsolutions(i.e.cellextracts,livecells;seepage17).Thisproprietaryphosphatasesubstrategeneratesaluminescentproduct(Em(nm)560nm)thatproducesstrongluminescenceuponinteractionwithphosphatase.Thekitprovidesalltheessentialcomponentswithouroptimized‘mixandread’assayprotocolthatiscompatiblewithHTSliquidhandlinginstruments.Ithasextremelyhighsensitivity,andcanbeusedfor the assays that require demanding sensitivity.

Description P/N : QtyChemiluminescentAPassaykit JQ6760 100 testsSeedescriptioninenzymedetection/AlkalinePhosphatasesection,page18.

Other Colorimetric & Fluorimetric Enzy-matic Substrates for Microplates assays

Peroxidase(HRP)andAlkalinephosphatase(AP)enzymatiquelabelsarewidelyusedinvariousbiologicalassays includingELISAs,aswellas in immunohistochemicaltechniquesandWesternblotanalyses.Following are effective ready to use substrates for colorimetric and fluorometricdetectionsinEIA.

Following is a selection of conventional and unique formulations of standardenzymaticsubstrates to fulfill therequirementsofyourmicroplatesassays.Belowareresearchcatalogquantities,pleaseasforHTSandbulkneeds.

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Other Colorimetric & Fluorimetric Enzymatic Substrates for Microplates assays

■UptimaTMBchromogenicsubstrateforHRPELISASolutions are optimized chromogenic substrates for peroxidase, designed for ELISA techniques, manual or automatic systems. They contain3,3’,5,5’-tetramethylbenzidine(TMB),hydrogeneperoxide(H2O2),andproprietarycatalyzingandstabilizingagents.

» Highest sensitivity» Reproduciblelots» Ready-to-use

Description P/N : QtyTMB,"Standard"solution UP664780 200mlThe original formulation, ideal for classic applications UP664781 500ml - Highest sensitivity UP664782 1L-Stability>24monthsat+4°C

TMB,"Check"solution UPS08170 100 mlRecommendedforroutineassays,tobettercontrolfailurein UPS08171 500mlreagentdistribution.Includesareddyethatdonotinterferewithreactionnorfinalreading.Highsensitivity;Stability>1yearat+4°C

TMB,"Aqueous"solution UPS08180 100 mlRecommendedinparticularfordiagnostickits UPS08181 200mlNon-hazardous,non-volatile,non-organic,non-toxic UPS08182 500ml(doesnotcontainDMF,DMSO) UPS08183 1L100%waterbasedformulationtomaximizethesafety(noregulationconcerns).Highestsensitivity;Stability>1yearat+4°C.

■ADHPHRPELISAAssayKitADHPprovideshighersensitivityinELISAthatchromogenicsubstrates.ThekitprovidesthesubstrateandthestabilizedPeroxidebufferinready-touseconvenientformatforHTSapplications.Thefluorescentsignalisreadat590nm(λexc\λem=530-571/590-600nm)andachievesdownfemtomolardetectionsensitivity.Itprovidesthereagentstoperform500ELISAassaysina96-wellformat.Theprotocolcanreadilybemodifiedtorunassaysina384-wellformat.

Description P/N : QtyADHPHRPELISAassaykit–ADHPbased HS6241 500testsContainsADHPsubstrateandPeroxideAssaybuffer(50ml)

■FluorimetricAPELISAAssaykitsSeeassaykits#JQ6730(Blue-MUP),#JQ6740Green-Red)insection"Apoptosis"page17.


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Other Colorimetric & Fluorimetric Enzyma-tic Substrates for Microplates assays

■SubstratesforhorseradishperoxidaseHRPsubstrates P/N : (a) Qty Format (c) Type (b) Comment

OPDtabletsof15mg 270861 50tabs Tabs Chromo. Eachtabcontains15mgofOPDforquickandeasypreparationofsubstratesolution

OPD,Ultrapurepowder 02673F 25g Pow. Chromo. (o-PhenyleneDiAmine)CAS:[95-54-5];MW:108.1 02673G 50g

ABTS,Liquidsubstrate UP732550 100 ml Soln Chromo. Ready-to-usesolution

ABTStablets 42387C 50tablets Tabs Chromo. Eachtabcontains10mgofABTSforquickandeasypreparationofsubstratesolution.

ABTS,Ultrapuregrade UP423876 1 g Pow. Chromo.CAS:[30931-67-0];MW:548.7 UP423877 10 g

TMBsolutionforELISA UP66478 Soln Chromo. Highestsensitivity.Seedescriptionpage64.

TMB,Ultrapurepowder UP15426D 1 g Pow. Chromo. (tetramethylBenzidine)CAS:[64285-73-0];MW:331.3 UP15426E 5g

DABsolution(50X) UP732320 500ml Soln Chromo. Ready-to-usesolutionlesstoxicformanipulationSuppliedasa50xconcentratewitha10xdilutionbuffer.

DABtabletsof5mg UP732310 50tabs Tabs Chromo. Eachtabcontains5mgofDABforquickandeasypreparationofsubstratesolution

DAB,Ultrapurepowder UP01012G 5g Pow. Chromo. (3.3'-DiAminoBenzidine)CAS:[868272-85-9];MW:360.1 UP01012H 10 g

ADHPHRPAssayKit. HS6241 500tests Kit Fluo. Page64.

ADHP,PureGradepowder FP-39423A 5mg Pow. Fluo. HighlyandstablefluorigenicsubstrateCAS:[119171-73-2];MW:257.2 FP-39423B 25mg λabs/λem.:563/587nm(10-Acetyl-3,7-Dihydroxyphenoxazine)

Resorufin,PureGrade FP-95432B 100 mg Pow. FluoCAS:[635-78-9];MW:213.04

Luminol,PureGradepowder FP-57578A Pow. Lum. λabs/λem.:355/413nm;EC:7650l/molxcmCAS:[521-31-3];MW:177.2

Luminol,Nasalt FP-CA9611 2.5g Pow. Lum. SeealsoUptiLightpage62.CAS:[20666-12-0];MW:199.1(a)allproductsareavailableasbulkformats.Pleaseinquire.(b)Chromo.:Chromogenic;Fluo.:Fluorigenic;Lum.:Lumigenic(c)Pow;:powder;Soln:Solutionmono-componentorbi-component(2Soln)Seealsosubstratesforreporterassays(B-galactosidase,...)page16.

■SubstratesforAlkalinePhosphateHRPsubstrates P/N : (a) Qty Format (c) Type (b) CommentpNPPELISAsolution BP7080 500tests Kit ContainsAssaybuffer,washbuffer,stopsolutionandAPIIAb.

pNPPtabsof30mg UP732500 100tabs Tabs Chromo Eachtabletcontains30mgofpNPPforquickandeasyUP732501 1000tabs preparationofsubstratesolution.

pNPPtabsof5mg UP89562G 100tabs Tabs Chromo Eachtabletcontains5mgofpNPPforquickandeasyUP89562F 1000tabs preparationofsubstratesolution.

pNPP,Ultrapurepowder UP89562C 25g Pow. Chromo. (p-NitroPhenylPhosphate).CAS:[4264-83-9];MW:301.3 UP89562D 100 g

MUPNasalt,UltraPuregrade FP-30045A 100 mg Pow. Fluo. (MethylUmbelliferylPhosphate).CAS:[22919-26-2];MW:277.1 FP-30045B 500mg Usedfordetectingphosphatasesinsolution.

FP-30045C 5g MaximumfluorescenceatpHvalueof>10.FP-30045D 10 g

MUPPlus,Nasalt FP-JQ6710 25mg Pow. Fluo.MaximumfluorescenceabovepH7.0,forcontinuousassays.AlsousedfortheassaysthatrequireacidicpHsuchasacidphosphatases.Seedescriptionpage18.

MUPFreeacid FP-24119A 100 mg Pow Fluo SeealsoMUPbasedAPassaykit#JQ6730page17.CAS:[3368-04-5];MW:256.1

FDP FP-72573A 5mg Pow. Fluo. (FluoresceinDiPhosphate).CAS:[217305-49-2];MW:560.4 SeealsoFDPbasedAPassay#JQ6740page17.

FDPELISAkit HT0790 1000 tests Kit Fluo. ContainsAssaybuffer,washbuffer,stopsolutionandAPIIAb.

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Fluorescent secondary reagents for ELISA (II Abs, Avidin)

■StreptavidinconjugatesStreptavidinconjugatesaresecondstepreagentsforstainingwithbiotinylatedantibodies.Interchimcoversawiderangeofwavelengths.HereisaselectionofgreatFluoProbesdyes,theFluoProbes782,682,647HandR-PEbeingthemostusefulinmicroplatesassays:

Wavelength(nm)

200 305 355 405 455 480 505 530 555 580 605 655 705 755

200 295 345 395 445 471 495 520 545 571 595 645 695 745

805

800λem.

λabs.

Description λexc.(nm) λem.(nm) P/N : QtyFluoProbes®782 783 800 FP-IS1810 1 mg

FluoProbes®682 690 709 FP-BE8050 1 mg

FluoProbes®647H 653 675 FP-CA5640 1 mg

FluoProbes®594A 601 627 FP-CA5620 1 mg

FluoProbes®565A 563 592 FP-CA5610 1 mg

R-Phycoerythrin 565 576 FP-77776A 1 mg

FluoProbes®547H 557 572 FP-CA5570 1 mg

FluoProbes® 488 493 518 FP-BA2221 1 mg

RelatedproductsOtherlabeled(strept)avidinconjugatesareavailablefromInterchim

Forotherwavelengths,pleasecontactusorseeinInterchimBioScienceInnovationscatalog.

References:RowellE.etal.,OpposingRolesfortheCyclin-DependentKinaseInhibitorp27kip1intheControlofCD4+TCellProliferationandEffectorFunction,TheJournalofImmunology,2005,174:3359-3368.

SeeFluoprobesdyesdescriptionspage67.


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■SecondaryAntibodiesconjugatesWeareofferingarangeofhighqualitysecondaryantibodiesconjugatedwithourfluorophorescalledFluoProbes.Showingsuperiorfluorescenceproperties, thesefluorophoresareanexcellentalternativetotheconventionaldyessuchCyaninesandotherdyes.FollowingareselectedfluorophorescompatibleusingcommonexitationsourcesandstandardfilterssetsuchasFITC/Cy2,TRITC/Cy3,TR/Cy5,andInfraRed,.

BenefitsofFluoProbes-antibodyconjugates

» Highbrightness:FluoProbes®dyesshowanenhancedfluorescencecomparedto othersimilarconjugates(veryhighSignal/Noiseratio)» Highphotostability:OurFluoProbesdiesaremorephotostablethanmostother conjugatesallowinglongerreading/scaning.» Color choice:FluoProbes® dyes range in color from green to infra-red.» Readytousesolution:nodissolutionstepandriskofcontamination.» Highspecies-specificity:Thesecondaryantibodieswereselectedtohavehighest specificities.Itisthenpossibletohaveveryspecies-specificsecondaryantibodies conjugatedwiththebestfluorochromes.

Fluorescent secondary reagents for ELISA (II Abs, Avidin)

Description λexc.(nm) λem.(nm) P/N : QtyGoatanti-MouseIgG(H+L)-FluoProbes®782 783 800 FP-BW7970 1 mg-FluoProbes®682 690 709 FP-BE7250 1 mg-FluoProbes®647HMinxRat,Hu,Bov,Hrs,RbsrProt 653 675 FP-SC4000 1 mg -FluoProbes®547HMinxRat,Hu,Bov,Hrs,RbsrProt 557 572 FP-SB4000 1 mg -FluoProbes®488MinxRat,Hu,Bov,Hrs,RbsrProt 493 518 FP-SA4000 1 mg

Goatanti-RabbitIgG(H+L)-FluoProbes®782 783 800 FP-BW7980 1 mg-FluoProbes®682 690 709 FP-BF1690 1 mg-FluoProbes®647HMinXBov,Ck,Gt,GP,Hms,Hrs,Hu,Ms,Rat,ShpSrProt 653 675 FP-SC5000 1 mg -FluoProbes®547HMinXBov,Ck,Gt,GP,Hms,Hrs,Hu,Ms,Rat,ShpSrProt 557 572 FP-SB5000 1 mg -FluoProbes®488MinXBov,Ck,Gt,GP,Hms,Hrs,Hu,Ms,Rat,ShpSrProt 493 518 FP-SA5000 1 mg

SeealloursecondaryantibodiesitemsinourcatalogBiosciencesInnovations.

Relatedproducts:PrimAb™:alargecollectionofprimaryantibodies.SearchourwebPrimAbsenginetoolathttp://www.interchim.com/interchim/PrimAb/search.cfm.Researchareasinclude:CellSignaling(ionic,cytokines,hormones),Apotosis/cellcycle,DNAreplications/transcription/Repair,post-translationalmodifications,Celladhesion,Enzymes,Membranestudy,Lipids,Metabolism,Angio/Histogenesis,Infectiousagents,biomarkersforCancer/Hypoxia,Cardiology,Neurosciences,Drugandresistance,Allergens.

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ImmunoAssay - Buffers & Saturating Agents

■StandardBuffersforImmunoassaysDescription P/N : QtyPBSpowderpack(makes10L) UP68723A 1packTBSpowderpack(makes20L) UP74004A 1packPBS(TrisBufferedSaline),20Xsolution N13761 1LPBStablets(1Tab.makes100mlof1Xsolution) 307150 100TabsPBSwithTween®,pH7.5 N13810 500mlTBS(TrisBufferedSaline),20Xsolution N14580 4LTBStablets(1Tab.=100mlof1Xsolution) GS3660 100Tabs

■ReadytouseBuffersforImmunoassaysThesepoweredblendssaveyourtimeandarehighproteomicsgradeforoptimalresultsinimmunodetections.Description P/N : QtyTBSwithNon-FatPowderedMilk3% GS4160 5pk(42g/1L)TBSwithBSA1% GS4170 5pk(22g/1L)TBSwithTween®0.05% GS4200 5pk(12.5g/1L)PBSwithNon-FatPowderedMilk3% GS4180 5pk(39.8g/1L)PBSwithBSA1% GS4190 5pk(19.8g/1L)PBSwithTween®0.05% GS4250 5pk(10.4g/1L)AntibodyDiluent(ReadytoUse) HH6690 125mlAbdilutedinthissolutioncanbestoredforupto18monthsat+4°C.

■SeaBlocksaturatingagent(nonmammalianserum)» Non-mammaliannaturepreventsinteractionswithimmunoreagents(i.e.mammalianantibodies)» Lowerbackground» Excellenttosaturatehighbindingsurfaces,andGlutaraldehydeactivatedAminepolystyrene (whenBSA,caseinandotheragentsaregoodbutnotexcellentorevenpoorblockers).Description P/N : QtySeaBlock(standard,excelsasablockerinELISA) UP40301A 500mlSeaBlock,serumfreeinPBS UPAP1370 500mlSeaBlock,serumfreeinTBS UPAP1380 500ml

■OthersaturatingagentsandBufferscomponentsDescription P/N : QtyBSApowder UPQ84170 100 gOurstandardgradeandeconomicBSA,ubiquitousformost UPQ84171 500gbiotechnologies,includingimmuno-saturations. UPQ84170 1kgBSA30%solution UP900100 50mlUsingthissolution,forgetthehassleofweightinganddissolving UP900101 500mlBSApowder(noagregates!).Savetimeandmoney!

PolymerisedBSA,30%solution BJ1440 50mlPolymerisedBSAimproveseveraldetectionsystems

BioBlockmembraneblockingagent(inPBS) N13660 1LAneconomicstandardblockerbasedoncasein,optimizedfor N13650 1L(inTBS)positivelychargednylonorPVDFmembranesinnucleicacidorproteinblottingapplications.

Non-fatMilk 768701 500gApopularbloquer

Gelatin N13360 100 gCAS[9000-70-8];Bloomnumber:240-270;pH(28°C):4.5-5.5; N13361 500gWater(KF):<12%;Viscosity:35-45mpa

Prionex®, 10% sterile solution 901770 100 mlBSAalternative,stronglyreducesunspecificbindingofproteintoplasticmicroplatewalls

Tween®20,pure 15874A 1LTween®20,20%solution,oxidantfree UP158740 5x10mlHighlypureandpackagedinsealedampulsunderargontoincreasetheaccuracyofimmunoassays.Note:afullrangeofothergradesofBSAandotheralbuminsarealsoavailable.


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Antibody Labeling Kits

Fordirectconjugationofprimaryantibodiesoryourbiomoleculesofinterest,Interchimprovidesalargerangeofbiotinylationandfluorescentagentsaswellcompletekitswithdesaltingtools.

Forgeneraluse,werecommendtouseNHS-,Maleimide,orHydrazide-activatedreagents.

Also,completebiotinylationkitsareproposedfornonexperiencedinvestigators,aswellasforconvenientlabeling(spinformat)whileavoidingtobuyseparate reagents.

■Microspinbiotinylationlabelingkits-NH2and-SHEfficientandconvenientbiotinylationreagent

» Quick:only1hour(/NH2)or3hours(/SH)togetconjugates» Easy:allprocessesinasinglefiltrationtube» Reliable:highrecoveryofconjugates,evenfor500µgofIgG!

Biotin Labeling Kits are primarily used for the preparation of biotin-labeled IgG forimmunoassays.Weofferkitswithaveryconvenient format :spinfilterswherereactionandwashes takeplace, thatareavailablewith2couplingstrategies,and for2samplesizes(50-200μgIgG,orca1mg).

ThekitBG767biotinylatesonamines, themoststandardstrategy. Itusesasuccinimidylesteractivatedbiotin, and containsall necessary reagents for labeling3 samplesof IgGantibody (10μg to 200μg). Itcanalsobeused tobiotinylateanyproteingreater thanMW50000Da.The labelingprocess issimple.JustaddtheNH2-reactivebiotintoIgGsolutiononafiltermembrane,andincubateat37ºCfor10min.Ontheaverage,5to8biotinmoleculesconjugatetoeachIgGmolecule.ExceedingbiotinmoleculescanberemovedusingaFiltrationtubesincludedinthiskit.

ThekitBT3591biotinylatesonsulfhydrylstoobtainorientedanddefinedbiotinylation.Itusesamaleimide-activatedbiotin.Featuresaresimilar tokitBG7670,except1/there isanadditionalstep tocreatea freesulhydrylinthoseprotein(IgG)thatdonothaveone,withoutlossofaffinity;2/maleimideincubationoccursat37ºCfor30min.

Description P/N : QtyBiotinylationkit-NH2 reactive BG7671 3lab.of50-250mg

BG7671 1lab.of10mg

Biotinylationkit-SHreactive BT3591 3lab.of50-250mg

Alsoavailable:biotinylationagentsasstandalonesulfoNHS-lc-Biotin UP54398A 100 mg

NHS-PEO4-Biotin UPR20279A 50mgPEOspacerconfershydrophilicityallowingtoreachhighercoupligratioandtoyieldmorebioactiveandstableconjugates.Itisalsoavailablewithextendedspacerlength(PEO12).

NHS-SS-Biotin UPS073A 100 mgcleavablespacer

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Description λexc.(nm) λem.(nm) P/N : QtyFluoProbes®782 783 800 FP-CA6070 1kit(5labelings)FluoProbes®682 690 709 FP-BE8280 1kit(5labelings)FluoProbes®647H 653 675 FP-BZ9610 1kit(5labelings)FluoProbes®547H 557 572 FP-BZ9600 1kit(5labelings)FluoProbes® 488 493 518 FP-BE3750 1kit(5labelings)

Forotherwavelengths,pleasecontactusorseeinInterchimBioScienceInnovationscatalog.

Antibody Labeling Kits

■FluoProbes®ProteinLabelingKitsEasyantibodydirectconjugationwiththebrilliantandphotostableFluoProbesdyes!

FluoProbes® labelingkitsaredesigned for theeasy-to-useandefficient labelingofproteinwithmolecularweightsgreater than25kD, includingespeciallyantibodies.Theyuseasuccinimidylesteroffluorescentlabelsthatformacovalentstablelinkage.Up100µgto1.5mgofprotein(IgG)canbelabeledina1h30procedure.TheyareavailablewithmanyofourFluoProbes®labels.Followingisalistofselectedandpopularones(otherson inquire).

SeeFluoprobesdyesdescriptionspage67.


Accessory tools


FPlyte Microplates

Microplateinnovationenablesleadingedgescreeningassays

» LongwavelengthUVmicroplates» Limitedwell-to-welllightcrosstalk» Improvedcellbindingefficiency

TheFPlytemicroplatesaremicroplatesfullycompatiblewithcommerciallyavailableplatereaders,roboticsampleprocessorsandautomatedliquidhandling systems. Theyareavailablein2formats,96-and384-wellsand3colors:

Theblackplateprovidestheall-absorbingbackgroundneededtominimisebackgroundinterferenceforsensitivefluorescencemeasurements.Theopaquewhiteplatemaximisesreflectivityenablingevenweaklyemittingluminescenceassaystoberoutinelyundertaken.Inaddition tooptimised luminescenceandfluorescencemeasurements theuniquedesignoffers improvedcellbindingefficiencyandallows theconvenienceofdirectmeasurementsonbottomreadingspectrophotometersandinvertedmicroscopes.

FPlytemicroplatesareidealforquantitativeassaysatexcitationwavelengthsinthelong-wavelengthUVareabetween325nm–425nm.Theyofferexcellentphotometricperformancedownto325nm(80%Tat325nm,100%Tat335nm).Wavelengths below 350 nm are particularly useful for a variety of fluorescence assays such as HNK-1 (λexc./em.: 325/380 nm), Thiguanine (λexc./em.:330/410nm)usingblackFPlytemicroplates,aswellasmanyabsorbanceassaysincludingVitaminA(325nm),retinolandretinylacetate(325nm),caspase(325nm),acidphosphatase(330nm)andhydroxyproline(335nm)usingwhiteFPlytemicroplates.

Description Colour P/N : Qty96-wellFPlyteMicroplate,standard Black FP-BA7991 50u

Black FP-BA7990 100 uWhite FP-BA7950 100 u

96-wellFPlyteblackwell,clearbottom Black FP-KT225A 50uBlack FP-KT225B 100 u

96-wellFPlyteMicroplate,TissueCultureTreated,withlids Black FP-BA8010 100 uWhite FP-BA7970 100 u

HiBind,96-wellFPlyteMicroplate Black FP-BA8000 100 uWhite FP-BA7960 100 u

Twister™HighThroughputScreeningPack,withlids,96-well Black FP-BA8020 80 uWhite FP-BA8030 80 u

384-wellFPlyteMicroplate,standard Black FP-BA8170 100 uWhite FP-BA8130 100 u

384-wellFPlyteMicroplate,TissueCultureTreated,withlids Black FP-BA8180 100 uWhite FP-BA8160 100 u

Relatedproducts:Sealfilmforfluorescentassays FP-CD5130 25mx78mm(1roll)

FP-CD5110 500mx78mm(1roll)FP-CD5150 125mmx78mm(100units)

Pleasecontactusforotherwavelengthsoffluorescentreferencestandards.

Technical tipFluoresceindetectionlimitofaninstrumentBeginwithahardweigh-outofatleast4-5mgandsolubilizein100mMsodiumborate(pH9.5).BorateistheNISTbufferused,butitcanbereplacedby50mMphosphate(pH9).Thedetectionlimitsmayvaryslightly.Tochecktheabsorbancespectrumandback-calculatetoconfirmtheconcentrationbyaknownextinctioncoefficientascalibratedagainsttheNISTstandard.CalculatetheconcentrationoffluoresceinstocksolutionbyC(M)=(Amax/extinctioncoefficient)xdilutionfold,thelightpathis1cm,Amaxat492+-5nm,extinctioncoefficientis78,000M-1 cm-1.Makeadilutionseriesintothesamebuffer,startinginthelownMrangeanddilutedown.Formoststandardcurves,triplicatemeasurementsateachconcentrationaresufficient.Butclosertothedetectionlimit,itisrecommandtotake8replicates.Thisisimportantfortheblanksample,aswell.WithdetectionlimitsusingZ-factoranalysis,aresult>or=-1isconsideredtobeadetectablesignal.TheequationfordeterminingZ-factorsis1-((3*Sample+3*StdDevBlank)/(Ave.readSample-Ave.readBlank)).Description P/N : QtyFluorescein,standardsolution,100nM(494/519nm) FP-DO6630 50mlFluorescein,standard(494/519nm) FP-19365A 1 g

Newapplicationsareunderdevelopment.Contactusforyour special needs.

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IMAplate Technology (Intelligent MultiFunctional Analysis)

IMAplate is a polystyrene plate of 96 "microcuves". It is used to simplify and accelerate the handling of reagents,AND tomakemeasures ofabsorbenciesinUV,visibleorInfraRed,offluorescenceorluminescence,thiswithanystandardreaderofmicroplate,whileyieldingsignalsatleastequalorsuperioronmicrovolumes(5µL)!

■Applications» 96-channelpipettingforliquidtransfer: ex.pipetteandempty96x5µLinonly10seconds!

» 96bottom-freemicro-cuvettearrayforUV,VISorIRspectroscopy : ex.assayDNA/RNAandproteinsonjust5µLwithyourusualmicroplatereader!

» 96microwellplateforparallelreactions,immuno-assaysandcellassays :ex.do96reactionschemicalorenzymaticonauniquesupport,miniaturizeELISAswithonly5μlofreagents,highersignalsandsavingtime!

PoweredbyInterchimincollaborationwithBertholdtechnologiesGmbh

AddedvalueofIMAplates:

» Miniaturization:savinglimitedsamples andcostlyreagents>3-20x/loweredprice per test » Transfer of liquid integrated to assays » High analysis throughput, even manually» Robust-highlyreproducible» Reducereactionstimes (solid-phase assays)» Increasesensitivity

Analysiswithamicroplatereader

TechnologieIMAplate

100µL/10mL minutes5µL/1mL seconds

Méthodeclassique/Microplaquesstandard

Description P/N : QtyIMAplate™StartKit DR9621 1Kit*IMAplate™(96µcuves)-white§ DR9611 5eaIMAplate™(96µcuves)-black§ DT5431 5eaIMAplate™(96µcuves)-yellow§ DT5441 5ea*contains5IMAplatesandReaderadapter§ Whiteplatearerecommendedforluminescencemeasurments,Blackforfluorescencemeasurements,andyellowplatesforUV-visspectrometryandsamplehandling.


Accessory tools


IMAPlate Microplates

■HowIMAPlatework?» Loading,un-loadingandwashesaresimplified,acceleratedandreliable: samplesandreagentsandbuffersareloadedsimultaneouslybycapillaryforce(a)(precisevolume),assayed,thendrawnawaybyanabsorbent paper(c)orbycentrifugation.ex.1plate/samplescanbewashedinjust10seconds,withoutmachine!» Microcuvesof5µLsaveup20fold(rare)samplesandany(costly)detectionreagents(exinELISA).» Reading(b):theopticalpathisperfectlydefined,andlongertothoseofstandardmicroplates! Hence detection sensitivity is superior.» Themicrocuveshavenobottom!Thusnoparasiteopticalabsorptiontakeplace,andyoucanworkinUV,IR...,withsuperiorsensitivity. You even can recover the samples (c).» Themicrocuveshaveageometrymorefavorableforimmunoenzymaticreactions(surface/volume3.8xsuperior),comparedwithwellsof standardmicroplates:hencekineticisspeededateachstep(exincubations2foldshorterinELISA).

IMAplatetechnologycombinesadvantageouslynotablyinELISA,andformultiplexedanalysis.

IMAplateofferasolutionatthesametimemoreflexible,quickerandcost-effective,when:» Samplesareinlimitedquantityorprecious, » Reagentsarecostly(caseofcommercialkits),» Severalanalysisareperformedoneachsample(multiplex),» Tospeedstepsandhandlingwithreliability.

Examples of applications particularly appropriate :» Serologicalanalysisofmanyanalytesinsmallanimalsserums» Multiplexscreening(pharma,cosmeto,vaccines)

(a) (b) (c)

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Microplates

■Integrateyourdifferentmethods/analysisonauniquesupport,flexible&efficient,withoutinstrumentinvestment!

SPECTROMETRYex :DNA/RNAdosage(260nm)

Proteinsdosage(UV280nm)

compoundInfraRedanalysis

LIQUIDHANDLINGex :Easyµcuveloading(10sec/capillaryforce,automatable)

Easyµcuveun-loading(10sec/absorbant,centrifugation)(automatable)

MicroCuvette:5µlvolume

Colorimetricassays-ABSex :ProteinsDosages:BCAssay,CooAssay

Biochemistry(clinical,IAA):TestsGlucose...

CellBiology:MTT,SOD,Thromboxanes...

FluorimetricCellAssay-FLex :AssayofProteins:LavaPepofDNA/RNA:Evagreen

CalciumFlux:Fluo-3,Fluo-8...

Cellviability:UptiBlue...

Toxicology:LDH,Grantoxilux...

(Chemi)LuminescentAssays-ECLex :ELISAavecHRP/UptiLightECL

CalciumFlux:Coelenterazine/aequaporin...

Cellviability:ATP/lum...

Reporterassay:Luciferase,Coelenterazine...

Homogenous-Phaseassays–CELLAssays

CommercialKitsReducecost/testby2-5fold

"homemade"AssaysReduceby3-20thecostofreagents

Solid-Phaseassays-ELISA

ELISACommercialKitsReducecost/testby2-5foldANDTIMEBY20-50%

"homemade"ELISA:TMB,UptiLightECL...Reduceby3-20thecostofbuffers,saturants,antibodies,enz.substrates

Othersystemsofmuliplexedanalysis(Luminex,SpotELISA,Micro-Arrays)

IMAplatetechnology

Reducetimesofincubation,transfer,andwashes,...

Reducethevolumes&costofraresamples&costlyreagentsby3-20X

Increasethesensitivityofdetection


Accessory tools


■Legals-TrademarksAcellafromCellTechnologiesCytonin,PeroxyGlow,andTACSfromTrevigenCyToxiLuxandGranToxiLuxfromOncoImmunDeepBlueCfromBioSignalPackardFluoProbes,PrimAbandUptiBluefromInterchimMUPplus,Phospholite,andRhod-4fromABDPMAfromBiotiumTwisterfromCaliper

■AntifadeKitforMicroplateWhen exposed to excitation light, fluorescence intensity of dyes decreases due to theirphotooxidationorotherphotoreactions.Thereareveryfewfluorescentdyesthatcompletelyresistphotobleaching.Frequently,whenasectionhasbeenscannedrepeatedlyunderstrongexcitationlight, dyes could lose significant fluorescence signal before visual evaluation or photographycanbeaccomplished.Forexamples, thephotobleachingoffluoresceins(suchasFITC-labeledantibodies) has become a major problem in fluorescencemicroscopy. In severe cases (suchasphycoprotein-labeledbioconjugates), a fluorescence imageof high resolution cannot evenbe takendue to theextremelyhighphotobleaching rate.TheAntifadeKit is to reduce thedyephotobleachingrate,givingresearcherslongerobservationtime.Thekitcontainsalltheessentialcomponentsthatcanbereadilyappliedtoimagingexperiments.Theyareallpremixedandready-to-usesolutions.Thiskitisdesignedformicroplateformat.

Description P/N : QtyAntifadeKitforMicroplate FP-CL0530 1 plate

Documents

Special Micro Plate as Says 2009