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WESTARandWESTAR-ONERev0120200212 Page1

WESTAR / WESTAR ONE


AboutusCyanagenisabiotechcompanylocatedinBologna,dedicatedtoresearch,development and production of reagents for molecular diagnostic since2003andoneoftheleadingcompaniesinthefieldofreagentsforWesternblottingandElisa.Themainproductlinesarefocusedonchemiluminescenceandfluorescentdyesforbiologicalanalysis,genomics,proteomicsandchemicalsensors.They are based on Cyanagen internationally patented technologies andachieveoutstandingperformanceintermsofsensitivityandstability.The products are extremely versatile and perfectly suited to the latestanalyticalinstrumentation.TheseproductsarealsoavailableasOEM.Cyanagens.r.l.hasacertifiedQualitySystem

CERTIFIED

All WESTAR and WESTAR-ONE substrates are protected byUS7803573,EP1962095,US7855287,EP1950207,US2012009603(A1), CA2742025, EP2405016, foreign equivalents and pendingpatents.


Productmanual

WESTARWESTAR-ONE

ECLsubstratesforWesternBlotting

WESTARandWESTAR-ONEAREINTENDEDFORRESEARCHUSEONLYANDSHALLNOTBEUSEDINANYCLINICALPROCEDURESORFORDIAGNOSTICPURPOSES.

www.cyanagen.com


Tableofcontents1. Introduction 5

Storage/expiryWESTARproductlineWESTAR-ONEproductline

2. Componentsandothermaterialsrequired 6KitcomponentsOtherrequiredsolutions

3. PerformSDS-PAGE 74. Preparetransfermembrane 75. Transfertomembrane 8

Constantvoltageorcurrentduringtransfer?6. Membranestaining(optional) 97. Blockingthemembrane 98. Antibodyincubation 10

SuggestedAbdilutions9. Chemiluminescentdetection

12Autoradiographyfilmvs.imagingdevices10. Troubleshooting 13

HighmembranebackgroundIrregularblackspotsNobandsorweakbandsNon-specificbandsWhitebandsor“ghostbands”Unevenorjaggedbands

11. Orderinginformation 16


1. IntroductionTheperoxidase-catalyzedoxidationofluminolanditsderivativesproducesaweakflashoflightat425nm.Theincorporationofanelectrontransfermediatorintothebuffer forces the flash signal into a glow and greatly improves the analyticalcharacteristicsofthereactionintermsofincreasedsignalintensityandduration.1,2Recentworks3÷6have shown that,byadditionofa suitableacylationcatalyst, afurtherlargeincreaseinlightoutputisobserved.

WESTAR and WESTAR-ONE detection reagents are non-isotopic,chemiluminescence substrates, designed for the chemiluminescent detection ofimmobilizedproteinsandimmobilizednucleicacidsconjugatedwithhorseradishperoxidase(HRP).WESTARandWESTAR-ONEareintendedforresearchuseonly,andshallnotbeusedinanyclinicalprocedures,orfordiagnosticpurposes.

References:1. Kricka,L.J.(2000)MethodsEnzymol.305,370-390.2. Heindl,D.andJosel,H.P.(1997)Non-radioactiveAnalysisofBiomolecules,258-261.Springer,Berlin.3. Marzocchi,E.,Grilli.S.,DellaCiana,L.,Prodi,L.,Roda,A.andMirasoli,M.,(2008)Anal.Biochem.,377,189-

194.4. Vdovenko,M.M.,DellaCiana,L.,Sakharov,I.Yu.,(2009)Anal.Biochemistry,392,54-58.5. Vdovenko,M.M.,DellaCiana,L.,Sakharov,I.Yu.,(2010)BiotechnologyJournal,5(8),886-90.6. Vdovenko,M.M.,Zubkov,A.V.,Kuznetsova,G.I.,DellaCiana,L.,Kuzmina,N.S.,Sakharov,I.Yu.,(2010)J

ImmunolMethods,362(1-2),127-130.

Storage/expiryOneyearatroomtemperature(18-25°C).


WESTARproductline

WESTARisCyanagenproductlineoftwo-componentchemiluminescentsubstratesforWesternblotting.WESTARproductsofferdifferentlevelsofsensitivityforyourWesternblotting,allowingfrompicogramtofemtogramdetectionrange.Ourproprietarytechnologyenablesfinetuningofsignalintensityinordertoobtainthe assay sensitivity and signal duration best suited to meet each experimentalneed.

WESTAR-ONEproductline

WESTAR-ONE is Cyanagen product line of one-component chemiluminescentsubstratesforWesternblotting.WESTAR-ONEproductsareavailablefordifferentlevelsofsensitivitytocoveralldetectionneedsfrompicogramstolowfemtograms.Our premixed solution based on a proprietary technology enables an increasedexperimentalconsistencyavoidingpipettingerrorsandpossiblecontaminations.

WESTAR SUN NOVA2.0

ANTARES

ETACULTRA2.0

SUPERNOVA HYPERNOVA

Signalintensity Standard Medium High VeryHigh UltraHigh ExtremelyHigh

Signalduration Medium Medium Extended Extended Short Short

Proteinabundance High High Medium Low UltraLow ExtremelyLow

WESTAR-ONE BASIC PLUS EXTREMESignalintensity Medium High UltraHighSignalduration Short Medium Short

Proteinabundance High Medium UltraLow


2. ComponentsandothermaterialsrequiredWESTAR-Kitcomponents

• SolutionA:Luminolderivative/enhancersolution(amberbottle)• SolutionB:Peroxidesolution(whitebottle)

WESTAR-ONE

• Luminolderivative/enhancer/peroxidesolution(amberbottle)Otherrequiredsolutions

Solution Preparation

RunningBuffer

For1Lof10xRunningBuffer(stock):• 30.3gTRIS(250mM)• 144.0gGlycine(1.9M)• 10.0gSDS(1%w/v)• Diluteto1Lwithdistilledwater

For1LofRunningBuffer:• 100mLof10xTransferBuffer• Diluteto1Lwithdistilledwater

TransferBuffer

For1Lof10xTransferBuffer(stock):• 30.3gTRIS(250mM)• 144.0gGlycine(1.9M)• Diluteto1Lwithdistilledwater

For1LofTransferBuffer:• 100mLof10xTransferBuffer• 200mLofmethanol• Diluteto1Lwithdistilledwater

TBS-TBuffer

For1Lof10´́TBSBuffer(stock):• 24.23gTRIS-HCl(20mM)• 80.06gNaCl(136mM)• Diluteto800mLwithdistilledwater• AddNaOH1MuntilpHisabout7.6• Diluteto1Lwithdistilledwater

For1LofTBS-TBuffer:• 100mlof10´́TBSBuffer• Whilestirringadd1mLTween-20• Diluteto1Lwithdistilledwater

BlockingBuffer

With5%non-fatdriedmilk:• 5gNon-fatdriedmilk• Dissolvein100ml1´́TBS-TBuffer

With5%BSA:• 5gBSA(CohnfractionV)• Dissolvein100ml1´́TBS-TBuffer

Ponceaustainingsolution

For100mLof10xPonceaustainingsolution(stock):• Dissolve0.5gPonceauSin1.0ml

glacialaceticacid• Diluteto100mlwithdistilledwater• Wrapbottlewithfoiltoprotect

solutionfromlight

For1LofPonceaustainingsolution:• 100mlof10xPonceauSstaining

solution• Diluteto100mlwithdistilledwater


3. PerformSDS-PAGE

I. PreparefreshRunningBuffer.II. Load the gels being sure to keep a

tight seal between the gel-cast andthegasket.

III. Pour the running buffer into themiddle of the gels and check forleaks.

IV. Pour the rest of the running bufferintothebottomoftherunningtank.

V. Removecombsanduseapipettetoclean away any unpolymerizedacrylamide.

VI. LoadaproperprestainedMWstandardinonelane.VII. Loadsamplesintotherestofthewellsandfillanyemptywellwithsamplebuffer.VIII. Runat90÷130Vconstantvoltageuntilthedyefrontreachesthebottomofthe

gel. If the current is too high band smiling and smearing (diffuse band) arecommonlyseeneffects.

4. PreparetransfermembraneIfusingnitrocellulosemembraneplace intodistilledwaterslowly,withoneedgeata45°angle.Ifinsertedtoo quickly into the water, air gets trapped andproteinwillnottransferontotheseareas.Oncewet,equilibratethemembraneinTransferBuffer for15min.If using PVDF membrane activate it with methanol for 30 seconds. Rinse withdistilledwaterandequilibrateinTransferBufferfor15min.

• Forproteins>15kDausemembraneporesize0.45mm• Forproteins<15kDausemembraneporesize0.2mm

NOTE: Low molecular weight proteins (< 15kDa) are sometimes transferredthroughnitrocellulosemembranes,thereforemaybenotvisibleontheblot.PVDFmembranehashigherproteinbindingcapacitythannitrocellulosemembraneandisrecommendedforbestdetectionsensitivity.


5. TransfertomembraneI. Wet four filter papers in TransferBuffer.

II. Assemble the transfer sandwich in atray large enough to hold the plastictransfer cassette. Fill with TransferBuffersothatthecassetteiscovered.

III. Placethefirstfoampadontotheblacksideofthetransfercassettethenplacetwopre-wettedfilterpapersonthetopofit.

IV. Place the gel and moisten its surfacewithTransferBuffer.

V. Place pre-wetted membrane directly on the top side of the gel, then gentlyremoveallairbubbles.Theproteinswilltransferassoonasthegelisplacedonthemembrane,itsrepositioningcangenerateasmearedimage.

VI. Placeanothertwopre-wettedfilterpaperoverthemembraneandremoveallairbubbles.

VII. Completetheassemblybyplacingthelastfoampad and locking the top half of the transfercassette.

VIII. Fill the transfer tank withTransferBufferandplacethetransfercassette.

IX. Puta frozencoolingunit into the transfer tankandsurrounditwithiceinapolystyrenebox.

X. Runthetransferwiththefollowingsettings:Wettransfer:80÷100Vfor30÷60min.Semi-drytransfer:15÷25Vfor20÷30min.

XI. Whentransferiscomplete,removethemembraneandmarkitsorientationbycuttingacorner.

XII. Washthemembranetwicewithdistilledwater.Constantvoltageorcurrentduringtransfer?Thebuffercompositionchangesassaltsareelutedfromthegels,resultinginanincreaseincurrentandadropinresistance.Atransferusingconstantcurrentleadstodecreaseinvoltageaswellasresistance(I=V/R).Therefore,theuseofconstantvoltage provides the best driving force during transfer. However, when currentreaches over 500mA in constant voltage setting cooling the gel is crucial forpreventingjouleheatinginthetank.


6. Membranestaining(optional)I. Stain themembranewithproteinsideupusingPonceaustainingsolution for5minutesatRTtochecktransferefficiency.

II. Rinse the membrane in distilled water untilproteinbandsaredistinct.

III. Scanthemembraneifdesired.IV. Completelydestainthemembranebyimmersingitfor10mininalargevolume

ofdistilledwater.V. Re-activatePVDFmembranewithmethanolthenwashinTBS-TBuffer.NOTE:

• ThebackgroundstainingtendstobehighwithsomedyeswhilePonceaustainingsolutiongivesaverycleanpattern.

• Re-activatePVDFmembraneafterstaining.• TheLODforPonceaustainingsolutionis250ngofprotein.

7. BlockingthemembraneI. Placethemembranewithproteinsideupintoafresh

traywithyourchoiceofBlockingBuffer.II. IncubatethemembraneinBlockingBufferfor30÷60

minutes with gentle agitation on a rocker/shaker. Amaximumblockingtimeof2hoursatRTshouldnotbeexceeded.Blocking for too longcanresult inantigenmaskingandlossofprotein.

III. RinsethemembranetwicewithTBS-TBuffer.

NOTE:Add3%non-fatdrymilkinTBS-TBufferwhendiluteAbtoreducenonspecificbindings.Milkcontainsmanyproteins,whichbindtothemembrane.So,aftertransfer,proteinscontainedinthemilkbindtothemembraneandfillalotofpotentialnonspecificsites.Afterthis,whenyouincubatewithyourantibody,itbindstotheantigenandhaslesspossibilitiesofnonspecificbinding.Ifyouare working with anti-phosphoproteins or with biotinylated

antibodiestheaddingofmilkisnotappropriate.Use5%BSAinstead.


8. AntibodyincubationI. Dilute the primary antibody in fresh TBS-TBuffer to the suggested primary

antibodydilution(seetablebelow).II. Incubatethemembranewithproteinsideupintheprimaryantibodysolutionfor

1to2hoursatRT.Toincreasesensitivity,tryanovernightincubationat4°Cwithagitationonarocker.MakesurethemembraneiscompletelycoveredwithTBS-TBufferwithprimaryAb.

III. Washthemembranewithproteinsideup4timesfor3to5minuteseachwithTBS-TBufferwithgentleagitationonashaker.AftereachwashingplacethemembraneonanewcleantraywithfreshTBS-Tbuffer.

IV. Dilute the secondary Ab in fresh TBS-T Buffer to the suggested secondaryantibodydilution(seetablebelow).

V. Incubate the membrane with protein side up for 30 minutes to 1 hour at RT.Increasingtheincubationtimeofthesecondaryantibodyusuallyleadstohigherbackground.

VI. Washthemembranewithproteinsideup4timesfor3to5minuteseachwithTBS-TBufferwithgentleagitationonashaker.AftereachwashingplacethemembraneonanewcleantraywithfreshTBS-Tbuffer.


IMPORTANT:OptimalAbdilutionsmayvarybetweendifferentapplications and depend on quality and affinity for the targetprotein. It iscrucialtooptimizebothprimaryandsecondaryAbdilutions for best results with high signal and low background.OptimalAbdilutionscanbedeterminedbyDot-Blotassay.

Product Suggestedantibodydilutions

WESTARSUNCod.XLS063WESTAR-ONEBASICCod.XLSU177

PrimaryAb1:100-1:5,000SecondaryAb1:1,000-1:15,000

WESTARNOVA2.0Cod.XLS071


WESTARANTARESCod.XLS142WESTAR-ONEPLUSCod.XLSU178


WESTARETACULTRA2.0Cod.XLS075 PrimaryAb1:5000-1:50,000SecondaryAb1:50,000-1:250,000

WESTARSUPERNOVACod.XLS3WESTAR-ONEEXTREMECod.XLSU180


WESTARHYPERNOVACod.XLS149 PrimaryAb1:10000-1:200,000SecondaryAb1:300,000-1:1,000,000


9. ChemiluminescentdetectionWESTAR:PrepareWESTAR working solution (WESTAR WS) by mixing properly eachreagentina1:1ratio.Forbestresults,prepareWSimmediatelybeforeuse.Donotcontaminatethesolutionswiththesamepipettetips.WESTAR-ONE:thesolutionisreadytouse.I. RemovethemembranefromitstrayofTBS-TBuffer,rinsethemembrane

twice with TBS-T Buffer, and keep it in TBS until the incubation withWESTARWS.

II. Use0.1mlofWESTARWS/WESTAR-ONEpercm2ofmembrane.Allowtheexcessbuffertorunofffromacorner.Donotletthemembranedryout.Justpipettethevolumerequireddirectlyontothemembranewithproteinsideupandincubatefor1,5minensuringthattheentiresurfaceiscovered.

III. Acquire thesignalwithautoradiography filmor imagingdevices.Foranunknownsignal,trytoexpose15s,30s,1minand5mintostartwith.

Autoradiographyfilmvs.imagingdevicesNowadays, Western Blotting is used either for absolute quantification (incombination with a calibration curve of the recombinant protein of knownconcentration)orforquantificationofsamplesrelativetoacontrolsample.Throughthedevelopmentofnewtechnologiesmost imagersofferawidedynamicrange(3÷5 orders of magnitude) generating a high-quality image compared with thelimitedlineardynamicrangeoffilm(1.5ordersofmagnitude).Thismeansthatispossibletoquantifybothstrongandweaksignalsonthesameblotwithreliableresults. Instead, on film strong signals get saturated resulting in a wrongquantitation.


10. Troubleshooting

Highmembranebackground

HighconcentrationofAb.FurtherdiluteprimaryandsecondaryAb.FollowsuggestedAbdilutions.Inefficient blocking. Increase Tween-20 in TBS-TBuffer(0.1%÷0.5%v/v).Use5%non-fatdriedmilkasblockingbufferifpossible.

Insufficientwashing.Increaseboththevolume,lengthandnumberofwashsteps.Alwaysusesufficientvolumestosubmersethemembrane.Primaryantibodyisnotspecificfortheproteinofinterest.Usemonospecificorantigenaffinitypurifiedantibodies.Alwaysincubateyourprimaryantibodyat4°Covernight and not at room temperature. Reduce NaCl in TBS-T Buffer(100mM÷350mM).UsemonospecificorantigenaffinitypurifiedAb.Non-specificbindingofsecondaryantibody.Confirmthesecondaryisspecificby omitting the primary and running a secondary only blot. If bands develop,chooseanalternativesecondaryantibody.Incompatibleblockingagent.Non-fatdrymilkcontainsendogenousbiotinandisincompatiblewithavidin/streptavidinsystems.Substitutewith5%BSA.Poorqualityofantibodies.Qualityandageofprimaryandsecondaryantibodymayleadtobackgroundproblems.Poorhandlingofmembrane.Besuretohandlethemembraneonlywithcleanplastictweezersandnon-powderedgloves.Contaminated buffer solutions. Check buffers for particulate or bacterialcontaminate.Replaceoldbuffers.

Irregularblackspots

Air bubble trapped in membrane. Remove airbubbles by gently rolling a clean pipette or a test-tubeduringsandwichassembling.Unevenlyhydratedmembrane.Makesurethatthemembrane is fully immersed during washes andantibodyincubations.

Contaminatedequipment.Proteinorpiecesofgelremainingontheunitmaysticktothemembrane.Antibodycangettrappedinthegel,andthenarewashedoutpoorly,resultinginintenselocalizedsignal.


Aggregationofblockingagent.Whenblockingagentispowderstiritovernightat4°Ctomakesureitiscompletelydissolved.Interactionofthemembranewithsampletray.Alwaysusecleanplastictraystoavoidanytypeofcross-reactionFormationofaggregatesinHRP-conjugate.Filtersecondaryantibodysolutionthrougha0.2μmfilter.Usefreshantibody.

Nobandsorweakbands

Excessive signal generated. The enzyme in thesystemdepletedthesubstrateandcausedthesignaltofadequickly.FurtherdilutesecondaryAb.Inefficient transfer. Ensure that there is goodcontactbetweenmembraneandgelduringsandwich

assembling.HighMWproteinmayrequiremoretimefortransfer.Reducevoltageortimeoftransferforlowmolecularweightproteins(<10kDa).Antibodiesmay have lost activity. Perform a Dot Blot. Follow manufacturer'srecommendedstorageandavoidfreeze/thawcycles.Incorrectsecondaryantibodyused.Confirmhostspecies/IgtypeofprimaryAb.Low protein-antibody binding. Reduce the number of washes to minimum.ReduceNaClinTBS-TBuffer(100mM÷350mM).Non-fatdrymilkmaymasksomeantigens.Decreaseblockingtime.DecreasemilkpercentageinBlockingBufferorsubstitutewith5%BSABlockingBuffer.Sodiumazidecontamination.MakesurebuffersdonotcontainsodiumazideasthiswillquenchHRPsignal.Contaminated stock solutions. Do not contaminate the chemiluminescentsubstratestocksolutionsusingthesamepipettetip.Usenewreagents.

Non-specificbands

Aggregation of analyte. Increase amount ofreducing agent to ensure complete reducing ofdisulfidebonds.SDSinterference.ThepresenceofSDSmayresultinthe development of unspecific bands caused by

antibodies binding to the charged SDS molecules associated with the proteins.Washthoroughlythemembraneaftertransferwithwater.Highproteinconcentration.Acommonlyseemeffectisthediffusionofproteinbands.Reducetheamountofproteininitiallyloaded.Primaryantibodyisnotspecificfortheproteinofinterest.Usemonospecificorantigenaffinitypurifiedantibodies.Alwaysincubateyourprimaryantibodyat4°C


overnight and not at room temperature. Reduce NaCl in TBS-T Buffer(100mM÷350mM).UsemonospecificorantigenaffinitypurifiedAb.Non-specificbindingofsecondaryantibody.Confirmthesecondaryisspecificby omitting the primary and running a secondary only blot. If bands develop,chooseanalternativesecondaryantibody.

Whitebandsor“ghostbands”

Excessivesignalgenerated.Excessiveantibodiesorloaded protein can cause high levels of localizedsignal.Thisresultsinrapidconsumptionofsubstrateatthispoint.Sincethereisnolightproductionafterthecompletionofthisreaction,whitebandsarethe

result.Tryfirsttofurtherdilutesecondaryantibody.

Unevenorjaggedbands

Unevengelrun.Loadallavailablewells.Emptywellscanbeloadedwithsamplebuffer.Voltage or current were too high duringelectrophoresis. Reduce voltage or current duringelectrophoresis.Effects of high salt in samples. Reduce NaCl

concentrationinTBS-TBuffer(100mM÷350mM).


11. SelectionGuide

Product Competitors

WESTARSUNCod.XLS063WESTAR-ONEBASICCod.XLSU177

AMERSHAM™ECL™-GEHEALTHCARE AMERSHAMECLSTART™-GEHEALTHCARE

PIERCE™ECL-THERMOSCIENTIFIC™ IMMOBILON®CLASSICO-MILLIPORE™

WESTARNOVA2.0Cod.XLS071

PIERCE™ECLPLUS-THERMOSCIENTIFIC™ IMMOBILON®CLASSICO-MILLIPORE™

WESTERNLIGHTNING™PLUS-PERKINELMER WESTERNBRIGHT™ECL-ADVANSTA

WESTARANTARESCod.XLS142WESTAR-ONEPLUSCod.XLSU178

CLARITY™-BIORAD SUPERSIGNAL™WESTDURA–THERMOSCIENTIFIC™

AMERSHAM™ECLPRIME™-GEHEALTHCARE SUPERSIGNAL™WESTPICOPLUS–THERMOSCIENTIFIC™

IMMOBILON®CRESCENDO-MILLIPORE™ WESTERNBRIGHT™QUANTUM™-ADVANSTA

WESTARETACULTRA2.0Cod.XLS075

SUPERSIGNAL™WESTDURA–THERMOSCIENTIFIC™ AMERSHAM™ECLPRIME™-GEHEALTHCARE

IMMOBILON®FORTE-MILLIPORE™ IMMOBILON®-MILLIPORE™

WESTERNLIGHTNING™PRO-PERKINELMER

WESTARSUPERNOVACod.XLS3WESTAR-ONEEXTREMECod.XLSU180

CLARITYMAX™-BIORAD SUPERSIGNAL™WESTFEMTO–THERMOSCIENTIFIC™

AMERSHAM™ECLSELECT™-GEHEALTHCARE WESTERNBRIGHT™SIRIUS™-ADVANSTA

WESTERNLIGHTNING™ULTRA-PERKINELMER

WESTARHYPERNOVACod.XLS149 NOCOMPETITORSATTHESAMEPERFORMANCELEVEL

Forfurtherinformation,visitwww.cyanagen.com

Fororders:[email protected]

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WESTAR / WESTAR ONE

westar … · western blotting, allowing from picogram to femtogram detection range. our...

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