Radiance PlusChemiluminescent substrate for the most sensitive Western blotting experiments

Long Protocol for Catalog Numbers

AC2102 RadiancePlusSample,sufficientfor200cm2 membrane

AC2103 RadiancePlus,150ml,sufficientfor1500cm2 membrane

Important InformationThe following instructions are for use with the Radiance Plus enhanced chemiluminescent substrate kit, catalog numbers AC2102 and AC2103. Please see the Kit Contents section for details.

Storage InformationThe Radiance Plus reagents are stable at room temperature for at least one year. For more information, see the Shipping and Storage Conditions section on page 3.

Warnings and Precautions•RadiancePlusisforresearchuseonly.•Alwaysweargloveswhenhandlingmembranesandreagents.•RefertoMSDSforadditionalsafetyinformation.•Theproductisguaranteedtobefreeofmanufacturerdefect,and

to function as described when the enclosed protocol is followed by properly trained personnel. Please see the Warranty section for more information.

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Table of ContentsSection Page

1. Kit Contents 3

2. Shipping and Storage Conditions 3

3. AdditionalMaterialsRequired 4

4. Background 4

5. Western Blotting 5

6. Overview of the Protocol for Chemiluminescent Western Blots 7

7. Quick Protocol 8

8. DetailedProtocol 9

9. TroubleshootingandFAQ 12

10. References 13

11. Related Products 13

12. Warranty 14

13. User Notes 14

1. Kit ContentsAC2102RadiancePlusChemiluminescentHRPSubstrate,Samplesize,sufficientfor 200 cm2 of membrane surface

•RadiancePlusLuminol/enhancersolution 7ml

•RadiancePlusPeroxideChemiluminescentDetection 7mlReagent

AC2103RadiancePlusChemiluminescentHRPSubstrate,Samplesize,sufficientfor 1000 cm2 of membrane surface

•RadiancePlusLuminol/enhancersolution 50ml

•RadiancePlusPeroxideChemiluminescentDetection 50mlReagent

2. Shipping and Storage ConditionsProduct may be shipped at ambient temperature. No extra temperature controlorinsulationisrequired.Allshippingmethods(groundandexpress) are acceptable.

TheRadiancePlusreagentsarestableatroomtemperature(upto+25°C)foratleastoneyear.Accidentalfreezingdoesnotsignificantlyaffecttheperformance,butmultiplefreeze-thawcyclesarenotrecommended.

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3. Additional Materials Required•ElectrophoresisapparatusandbuffersforSDS-PAGE

•Tankandbuffersforelectrophoretictransferofproteinsfromgeltomembrane

•NitrocelluloseorPVDFmembrane,cuttosizeofgel.AllmembraneproductsavailablefromAzure(seetheRelatedProductssection)arecompatible with Radiance Plus.

•Washingbuffer(PBS-TorTBS-T).Forbestresults,useAzureFluorescentBlotWashingBuffer(seetheRelatedProductssection)

•Blockingbuffer

•Primaryantibodycompatiblewithyourapplication

•Secondaryantibody,conjugatedtoHorseradishperoxidase(HRP)correspondingtoyourprimaryantibody(seetheRelatedProductssection)

•CCD-baseddetectionsystem,orfilm(seetheRelatedProductssection)

4. BackgroundRadiancePlusisahorseradishperoxidase(HRP)substratespeciallydeveloped for Western blotting detection of very low abundance proteins. RadiancePlusproducesastrong,long-livedsignal,which,combinedwith very low background levels, allows for long exposure times enabling thedetectionoflow-abundanceproteins.Additionally,thesignalfromRadiance Plus is linear with respect to protein amount over a broad range ofconcentrations,allowingtheusertoaccuratelyquantifyproteinbands.RadiancePlusismostsuitablefordetectionoflow-abundanceproteinsor in situations when amounts of available primary antibodies are very limited and high dilution factors are desired, or when primary antibodies have relatively low binding constants. Radiance Plus is also compatible withX-rayfilmdetection,thoughthelimiteddynamicrangeoffilmwillmakeresultingdatalessquantitative.RadiancePlusalsoproducesachemifluorescentsignalthatcanbedetectedwithafluorescenceimagingsystem using appropriate excitation and emission settings.

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5. Western BlottingWestern blotting is a protein analysis tool that has become commonplacein a molecular biology and protein chemistry laboratory. The principle ofchemiluminescent Western blotting is shown in Figure 1. The general protocol, including the role of Radiance Plus, can be seen on page 7. Proteins are separated by size via electrophoresis, and then transferred electrophoretically from the gel to a membrane support, usually nitrocelluloseorPVDF.Thismembranecontainingthetransferredproteinsis commonly referred to as a blot. The location of a protein of interest is detected on the blot by applying the primary antibody, which binds to the protein. The primary antibody bound to the blot is then visualized using a secondary antibody that binds to the primary antibody. The secondary antibody is labeled in some way to make it detectable, such as with a radioactive isotope or an enzyme that can be detected by its activity.

Figure 1. The principle of chemiluminescent Western blotting.

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Figure 2. Chemiluminescence of luminol.

NH2

NH

NH

Luminol

HRP + H2O2

NH2

+ N2 + light

Since1988,enhancedchemiluminescenceorECL(1)hasbecomeoneofthemostcommondetectionmethodsinWesternblotting(2).Inthismethod,thesecondaryantibodyisconjugatedtotheenzymeHorseradishperoxidase(1,2).Onceboundtothemembrane,thesecondaryantibodyis detected by incubating the blot with a solution containing an HRPsubstratethatgeneratesalight-emittingproductafterreactionwithHRP(Figures1,2).ThechemiluminescentsignalcanbedetectedbyexposingtheblottoX-rayfilm,orbyimagingwithaCCDcamera.

Radiance Plus is an enhanced chemiluminescent substrate speciallyformulated to achieve high sensitivity. Radiance Plus produces abright signal with very low background for femtomolar detection levels.Additionally, the Radiance Plus signal is long lasting, which combined withthelowbackground,allowslong-termexposurestodetectlow-abundance proteins.

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6. Overview of the Protocol for Chemiluminescent Western Blots

Transferproteins from gel to membrane

Blocktomasknonspecificproteinbindingsitesonmembrane

Primary antibodybinds to protein of interest

Washto remove excess antibody

Electrophoresis to separate proteins in sample

Secondary antibodybinds to primary antibody

Washto remove excess antibody

Substrate (Radiance Plus)substrate reacts with HRP bound to secondary antibody

to create luminescent signal

ImagedetectluminescentsignalwithCCDcameraorfilmorchemifluorescentsignalwithfluorescenceimager

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7. Quick ProtocolFor additional information, see the detailed protocol which follows.

Step User Notes

1. Prepare your protein blot

2. Block membrane for 1 hour at room temperature(RT)

3. Incubate blot with primary antibody for one hour at RT with gentle agitation

4. Wash blot:•1xquickly•1x15min,with0.7ml/cm2 membrane•3x5min,withatleast0.3ml/cm2

membrane each time

5. Incubate blot with secondary antibody for one hour at RT with gentle agitation

6. Wash blot:•3x5min,withatleast0.3ml/cm2

membrane each time

7.MixRadiancePluscomponents1:1toobtain0.1ml/cm2andplaceonblotfor2 minutes

8.Drainexcessreagent

9.CoverdampblotwithplasticwrapandimagewithCCDcameraorbyexposuretoX-rayfilmorwithfluorescenceimaging system

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8. Detailed Protocol

Step Notes

1. Prepare a protein blot

1.1. Separate the protein sample(s)viaelectrophoresis

1.2. Transfer proteins to membrane

•Prewetmembraneintransferbuffer,andassemble transfer sandwich according to tank manufacturer’s instructions.

•Dot-blotsorslotblotscan also be detected with Radiance Plus.

•Anyelectrophoresissystemandbuffer,suchasLaemmli,iscompatiblewithRadiancePlus.

•Awetortanktransfermethodispreferred,thoughsemi-drymethodsshouldalsobecompatible.WehavefoundthatthebuffersystemdevelopedbyBoltetall(3)workswell.

•BothnitrocelluloseandPVDFmembranesarecompatible with Radiance Plus.

•IfusingPVDF,firstwetmembranewitha1minincubationin100%MeOHfollowedbywaterfor~5minandthentransferbufferfor5-10min.

•Forslotblotapplications,nitrocelluloseismuchmoreconvenientthanPVDFbecauseitismoredifficulttoavoidbubbleswithPVDF.

2. Block membrane

•Incubatetheblotinablockingbufferwithgentleagitation for 1 hour at room temperature(RT).Use0.2to0.5mlofblockingbufferper cm2 of blot to provide adequateblocking.

•Blockingmasksnon-specificproteinbindingsites on the membrane, reducing background andincreasingthespecificityofbindingoftheprimary antibody to the protein of interest.

•Theoptimalblockingbufferwilldependinpart on the nature of the antigen of interest, andonthequalityoftheprimaryantibody.Commonblockingagentsincludingnon-fatdrymilk have been found to be compatible with Radiance Plus.

•10to20mlisusuallysufficientforatypical7x9cmmini-blot.

3. Incubate blot with primary antibody

•Diluteprimaryantibodyinblockingbuffer.

•Incubateblotwithprimaryantibody solution for 1 hour at RT with gentle agitation.

•Optimalprimaryantibodydilutionsmustbedetermined empirically.

•ForCCDimaging,werecommendprimaryantibody dilutions from 1:1000 to 1:10,000. A good initial dilution is 1:5000.

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8. Detailed Protocol, continued

Step Notes

3. Incubate blot with primary antibody, continued

•Iftheblotwillbeimagedonfilm,use2–5xlessprimaryantibodythanforCCDimaging.For example, if 1:1000 dilution of the primary antibodywasoptimalforCCDdetection,1:5000issuitableforfilmdetection.

•Antibodycanbeaddedtoadishandplacedonashaker,orasmallervolume(5-10ml)canbe used by sealing the blot into a bag and placing it on a rotary or rocking platform.

4. Wash blot to remove excess primary antibody

•1xquickly•1x15min,with0.7ml/cm

membrane•3x5min,withatleast0.3ml/cm2 membrane each time.

•Forbestresults,useAzureFluorescentBlotWashingBuffer(AC2113)whichisoptimizedforchemiluminescentaswellasfluorescentblots.PBS-TorTBS-Tarealsocompatiblewith Radiance Plus.

•Werecommendwashingorrockingblotsina clean dish on a shaker to provide gentle agitation.

•Forexample,astandard7x9membranerequires:~50mlofwashingsolutionforthe15min wash; and ~20 ml of washing solution for 5 min washes.

5. Incubate blot with secondary antibody

•Dilutesecondaryantibodyinblockingbuffer.

•Incubateblotwithsecondary antibody solution for 1 hour at RT with gentle agitation.

•Optimalsecondaryantibodydilutionsmustbedetermined empirically.

•Werecommendsecondaryantibodydilutionsof 1:5,000 to 1:20,000. A good initial dilution is 1:10,000.

•Iftheblotwillbeimagedonfilm,use2–5xlesssecondaryantibodythanforCCDimaging.1:50,000 dilution is a good starting point for filmdetection.

•Seealsonotesforstep3.

6. Wash blot to remove excess secondary antibody

•3x5min,withatleast0.3ml/cm2 membrane each time.

•Seenotesforstep4.

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8. Detailed Protocol, continued

Step Notes

7. Incubate blot with Radiance Plus

•Mixcomponents1and2ina1:1ratioinsufficientamounts to obtain at least 0.1ml/cm2 of the blot and add to the blot.

•Itisbettertopreparetheworkingsolutionjustbeforeuse. However, mixed reagent is stable for several hours at RT.

•Allowsubstratetoreactwith blot for 2 minutes.

•Becarefulnottotouchorputpressureontheblotasthiscanresultinnon-specificbackground.

•Useonlyplasticforceps,notmetal;metalforceps damage the blocked surface, creating new adsorption sites. Also, traces of metal mayactasacatalystfornon-enzymaticsubstrate oxidation, resulting in very high background.

•Theminimalamountofworkingreagentis0.1ml/cm2.Forexample,fora7x9cmblot,thisminimalvolumeis7x9x0.1=6.3ml.

•Ifusingtheminimalamountofworkingreagent, incubation may be done without agitation.Makesurethemembranesurfaceislevelsoadequatereagentisheldbysurfacetension.

•Incubationmayalsobedonewithgentleagitationinatrayjustslightlylargerthanthemembrane. Increase the reagent volume as necessary to ensure the membrane is adequatelycoveredwithreagent.

8. Drain excess reagent

•Removeexcesssubstrateviacapillaryactionby touching a KimWipe® or other absorbent material to the edge of the blot.

9. Image blot

•Whileblotisdamp,coverwith transparent plastic wrap and either place blot inCCDimager,orexposeblottofilm.

•Ifyouaregoingtousefluorescentimaging,incubation time with the substrate may be increased to 5 to 10 minutes.

•Werecommendtryingthreeexposures;30sec,2 min, and 5 min.

•Theblotcanbeimagedandre-imagedforseveral hours; for medium intensity bands about 60% of the initial signal will remain after 60 minutes, and substantial signal will remain after8-10hours.

•Excitationrangeisfrom430nmto490nmandcan be done with most blue light sources. Optimal emission range is from 500nm to 530nm.

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Problem Possible SoutionsHigh background •Reduceprimaryantibodyconcentrationbyincreasing

the dilution factor.•Tryadifferentblockingbuffer.•Tryashorterexposuretime.•Increasewashingtime.

No or low signal •Checkthatcorrectprimaryantibodyused.•Checkthatsecondaryantibodyrecognizesprimary(forexampleiftheprimaryisarabbitantibody,thatthesecondaryisgoat-anti-rabbit).

White spots withinbands

•Improvetransfer,makingsuretoremoveanybubblesbetween the gel and the membrane.

Speckled background •Filtersecondaryantibody.•Filterblockingandwashingbuffers.•Ensurethatthelaboratoryenvironmentisclean,to

minimize dust, debris or any other particles that might come in contact with the blot. Cover the dish during incubation or washing steps.

•Usenon-powderedgloves,orswitchtoadifferentkindofgloves.Werecommendpowder-freenitrilegloves or polyethylene gloves.

9. Troubleshooting & FAQWesternblottingcanrequiresubstantialoptimizationduetothemultiplesteps involved. The correct amount of protein to load on the gel and thebest dilutions of primary and secondary antibodies must be determinedempirically.Somecommonquestionsareaddressedbelow:

8. Detailed Protocol, continued

Step Notes

9. Image blot, continued

•Removeexcessreagentby blotting the membrane againstcleanfilterpaperortissue.

•Productsofthesubstrateoxidationduringthereaction with HRP are insoluble in water and will remain adsorbed to the membrane.

•Membranemaybeimagedwhilestilldamp,orit can be dried completely. The best imaging conditions should be determined by the user depending on the imaging instrument.

10. References1.ThorpeGH,KrickaLJ,MoseleySB,WhiteheadTP,Phenolsasenhancersofthechemiluminescenthorseradishperoxidase-luminolhydrogenperoxidereaction:applicationinluminescence-monitored enzyme immunoassays. Clin Chem. 1985Aug;31(8):1335-41.

2.LeongMM,FoxGR.,Enhancementofluminol-basedimmunodotandWestern blotting assays by iodophenol. Anal Biochem.1988Jul;172(1):145-50.

3.BoltM.W.,MahoneyP.A,High-efficiencyblottingofproteinsofdiversesizesfollowingsodiumdodecylsulfate-polyacrylamidegelelectophoresis.AnalBiochem.1997May1;247(2):185-192.

11. Related ProductsCatalog Number Product Size

AC2113 AzureFluorescentBlotWashingBuffer 500ml

AC2114 Goat-anti-rabbitHRP-conjugated 500μl secondary antibody

AC2115 Goat-anti-mouseHRP-conjugated 500μl secondary antibody

AC2105 LowFluorescenceWesternMembrane 10sheets (PVDF)7x9cm

AC2106 NitrocelluloseTransferMembrane 10sheets 0.45μm7x9cm

AC2107 NitrocelluloseTransferMembrane 10sheets 0.22μm7x9cm

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12. WarrantyThis product is warranted to be free of defects of material or workmanship,andtoperformasdescribedinthepublishedspecificationswhen stored according to the documentation included with the product, and used according to the accompanying instruction manual by appropriately trained personnel. If the product is found to have a defectuponfirstuseandwithin30daysofshipment,theproductmaybe replaced. This warranty extends only to the original purchaser of the product. There is no obligation to replace the product as a result of misuse, improper storage, or negligence of the buyer.

13. User Notes

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