AntibodiesOnline/STAT6 ELISA Kit (Signal Transducer and Activator of Transcription 6, Interleukin4

• Siteandsignalpathway-specific
• Invitrodetectionofadherentcellculture
• Nosamplelysisneeded
• CompatiblewithastandardELISAplatereader
• FasterresultsthanwithELISA
• Adaptableforhigh-throughputscreeninganddrugdiscovery

• Distilledordeionizedwater
• 100 mLand1litergraduatedcylinders
• Tubestopreparesampledilutions
• ProteaseandPhosphataseinhibitors
• Precisionpipettestodeliver2 μLto1 mLvolumes
• Adjustable1-25 mLpipettesforreagentpreparation
• Benchtoprockerorshaker
• Microplatereadercapableofmeasuringabsorbanceat450nm

1. Seed10,000-30,000cellsintoeachwellandincubateovernight.
2. Applyvarioustreatment,inhibitorsoractivatorsaccordingtomanufacturesinstructions.
3. Add100 μLofFixingSolutionintoeachwellandincubatefor20 minatRTwithshaking.
4. Add200 μLofprepared1XQuenchingBufferandincubate20 minatRT.
5. Add200 μLofBlockingSolutionandincubatefor1hat37 °C.
6. Add50 μLof1Xanti-phospho-proteinspecificantibodyoranti-pan-proteinspecificantibodytoeachwellandincubatefor2hatRT.
7. Add50 μLofprepared1XHRP-Anti-RabbitorMouseIgGandincubatefor1hatRT.
8. Add100 μLofTMBOne-StepSubstrateReagenttoeachwell.
9. Incubate30 minatRT.
10. Add50 μLofStopSolutiontoeachwell.
11. Readat450nmimmediately.

NOTE:Thawallreagentstoroomtemperatureimmediatelybeforeuse.Ifwashbufferscontainvisiblecrystals,warmtoroomtemperatureandmixgentlyuntildissolved.
NOTE:Brieflycentrifuge(~1,000g)ITEMSG,H,andIbeforeopeningtoensuremaximumrecovery.
Formoreinformationlookatthepicture.

NOTE:ALLincubationsandwashstepsmustbeperformedundergentlerockingorrotation(~1-2cycles/sec).
1.Designyourexperiment.Forexample,seeFigure2below.
OPTIONAL:IfseedingHUVECs,HMEC-1orotherlooselyattachedcells,coattheUncoated96-WellMicroplate(ITEMA)byadding100µLpoly-L-Lysine(RecommendedSigmaAldrich,Cat#:P4832)intoeachwellandthenfollowmanufacturersinstructions.Apre-coatedCellBIND®microplateorotherpoly-lysinetreatedtissuecultureplatemaybeusedinplaceofItemA.
2.Seed100µLof30,000cellsintoeachwelloftheUncoated96-WellMicroplate(ITEMA)providedandincubateovernightat37°Cwith5%CO2.
NOTE:Theoptimalcellnumberusedwillvaryonthecelllineandtherelativeamountofproteinphosphorylation.Moreorlesscellsmaybeusedbutthismustbedeterminedempirically.
NOTE:Thecellscanbestarved~4-24hours(dependingoncellline)priortotreatmentwithinhibitorsoractivators.
3.Applyvarioustreatments,inhibitors(suchassiRNAorchemicals)oractivatorsaccordingtomanufacturersinstructionsandincubateforthedesiredtimepoints.
NOTE:Itisrecommendedtodissolveinhibitorsoractivatorsintoserum-freecellculturemediumbeforetreatingthecells(unlessotherwisestatedinthemanufacturersinstructions.)
4.Discardthecellculturemediumbyflippingthemicroplateupsidedownandgentlytappingthebottomofthemicroplateoverasink.
5.Washbypipetting200µLoftheprepared1XWashBufferA(ITEMB)intoeachwell.Discardthewashbuffer(sameasstep4)andwash2moretimesforatotalof3washesusingfreshwashbuffereachtime.Afterthefinalwash,gentlyblotthemicroplateontoapapertoweltoremoveanyexcess/remainingbuffer.
NOTE:Toavoidcellloss,donotpipettedirectlyontothecells.Instead,gentlydispensetheliquiddownthewallofcellculturewells.Alsoavoidtheuseofvacuumsuctionortooforcefullytappingthemicroplatewhendiscardinganysolution.
6.Add100µLofFixingSolution(ITEMD)intoeachwellandincubatefor20minutesatroomtemperature.
NOTE:Thefixingsolutionisusedtopermeabilizethecells.
7.Repeatwashstep5.
8.Add200µLoftheprepared1XQuenchingBuffer(ITEME)intoeachwellandincubate20minutesatroomtemperature.
NOTE:Thequenchingbufferisusedtominimizethebackgroundresponse.
9.Wash4timeswith1XWashBufferA.
10.Add200µLoftheprepared1XBlockingBuffer(ITEMF)intoeachwellandincubatefor1hourat37°C.
11.Wash3timeswiththeprepared1XWashBufferB(ITEMC).
NOTE:Ifneeded,themicroplatemaybestoredat-80°Cforseveraldaysafterthiswash.
12.Add50µLoftheprepared1Xprimaryantibody(ITEMGorH)intoeachcorrespondingwellandincubatefor2hoursatroomtemperature.
13.Wash4timeswith1XWashBufferB.
14.Add50µLoftheprepared1XHRPConjugatedsecondaryantibody(ITEMI-1orI-2)intoeachwellandincubatefor1houratroomtemperature.
NOTE:ItemI-1isthesecondaryantibodyforItemG(primaryantibody).ItemI-2isthesecondaryantibodyforItemH(primaryantibody).
15.Wash4timeswith1XWashBufferB.
16.Add100µLoftheTMBSubstrate(ITEMJ)intoeachwellandincubatefor30minutesatroomtemperatureinthedark.
17.Add50µLoftheStopSolution(ITEMK)intoeachwell.Readat450nmimmediately.