Preparation
1.SamplePreparationandStorage
Storesamplestobeassayedwithin24hoursat2-8°C.Forlong-termstorage,aliquotandfreezesamplesat-20°C.Avoidrepeatedfreeze-thawcycles.
Cellculturesupernates:Removeparticulatesbycentrifugation,assayimmediatelyoraliquotandstoresamples
at-20°C.
Serum:Allowtheserumtoclotinaserumseparatortube(about4hours)atroomtemperature.Centrifuge
atapproximately1000Xgfor15min.Analyzetheserumimmediatelyoraliquotandstoresamplesat-20°C.
Plasma:Collectplasmausingheparin,EDTAorcitrateasananticoagulant.Centrifugefor15minat1500xgwithin30minofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.
2.SampleDilutionGuideline
Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.
Hightargetproteinconcentration(20-200ng/ml).Theworkingdilutionis1:100.i.e.Add1μlsampleinto99μlsamplediluentbuffer.
Mediumtargetproteinconcentration(2-20ng/ml).Theworkingdilutionis1:10.i.e.Add10μlsampleinto90μlsamplediluentbuffer.
Lowtargetproteinconcentration(31.2-2000pg/ml).Theworkingdilutionis1:2.i.e.Add50μlsampleto50μlsamplediluentbuffer.
Verylowtargetproteinconcentration(≤31.2pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.
3.ReagentPreparationandStorage
A.ReconstitutionofthehumanBDNFstandard:BDNFstandardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofBDNFstandard(10ngpertube)areincludedineachkit.Useonetubeforeachexperiment.
a.10,000pg/mlofhumanBDNFstandardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.
b.2000pg/mlofhumanBDNFstandardsolution:Add0.2mloftheabove10ng/mlBDNFstandardsolutioninto0.8mlsamplediluentbufferandmixthoroughly.
c.1000pg/ml→31.2pg/mlofhumanBDNFstandardsolutions:Label6Eppendorftubeswith1000pg/ml,500pg/ml,250pg/ml,125pg/ml,62.5pg/ml,31.2pg/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove2000pg/mlBDNFstandardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.
Note:Thestandardsolutionsarebestusedwithin2hours.The10ng/mlstandardsolutionshouldbestoredat4°Cforupto12hours,orat-20°Cforupto48hours.Avoidrepeatedfreeze-thawcycles.
- Preparationofbiotinylatedanti-humanBDNFantibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.
a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)
b.Biotinylatedanti-humanBDNFantibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanBDNFantibodyto99μlantibodydiluentbuffer.)
C.PreparationofAvidin-Biotin-PeroxidaseComplex(ABC)workingsolution:Thesolutionshouldbepreparednomorethan1hourpriortotheexperiment.
a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)
b.Avidin-Biotin-PeroxidaseComplex(ABC)shouldbedilutedin1:100withtheABCdilutionbufferandmixedthoroughly.(i.e.Add1μlABCto99μlABCdiluentbuffer.)
AssayProcedure
TheABCworkingsolutionandTMBcolordevelopingagentmustbekeptwarmat37°Cfor30minbeforeuse.Whendilutingsamplesandreagents,theymustbemixedcompletelyandevenly.StandardBDNFdetectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofBDNFamountinsamples.
1.Aliquot0.1mlperwellofthe2000pg/ml,1000pg/ml,500pg/ml,250pg/ml,125pg/ml,62.5pg/ml,31.2pg/mlhumanBDNFstandardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernates,serumorplasma(heparin,EDTA,citrate)toeachemptywell.See“SampleDilutionGuideline”abovefordetails.ItisrecommendedthateachhumanBDNFstandardsolutionandeachsamplebemeasuredinduplicate.
2.Sealtheplatewiththecoverandincubateat37°Cfor90min.
3.Removethecover,discardplatecontent,andblottheplateontopapertowelsorotherabsorbentmaterial.DoNOTletthewellscompletelydryatanytime.
4.Add0.1mlofbiotinylatedanti-humanBDNFantibodyworkingsolutionintoeachwellandincubatetheplateat37°Cfor60min.
5.Washplate3timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor1min.Discardthewashingbufferandblottheplateontopapertowelsorotherabsorbentmaterial.(PlateWashingMethod:Discardthesolutionintheplatewithouttouchingthesidewalls.Blottheplateontopapertowelsorotherabsorbentmaterial.Soakeachwellwithatleast0.3mlPBSorTBSbufferfor1~2minutes.RepeatthisprocesstwoadditionaltimesforatotalofTHREEwashes.Note:Forautomatedwashing,aspirateallwellsandwashTHREEtimeswithPBSorTBSbuffer,overfillingwellswithPBSorTBSbuffer.Blottheplateontopapertowelsorotherabsorbentmaterial.)
6.Add0.1mlofpreparedABCworkingsolutionintoeachwellandincubatetheplateat37°Cfor30min.
7.Washplate5timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor1-2min.Discardthewashingbufferandblottheplateontopapertowelsorotherabsorbentmaterial.(SeeStep5forplatewashingmethod).
8.Add90μlofpreparedTMBcolordevelopingagentintoeachwellandincubateplateat37°Cindarkfor20-25min(Note:Forreferenceonly,theoptimalincubationtimeshouldbedeterminedbyenduser.AndtheshadesofbluecanbeseeninthewellswiththefourmostconcentratedhumanBDNFstandardsolutions;theotherwellsshownoobviouscolor).
