| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy ReporterGeneEnzymes |
| Related | TagEnzymes |
1.PrepareassaymixforOne96-wellplate:
1.1 Prepareresorufinβ-D-galactosidesubstratestocksolution(200X):Add50µLofDMSO(ComponentE)intothevialofresorufinβ-D-galactosidase(ComponentA)tomakestocksolution(200X).
Note:25µLofresorufinβ-D-galactosidasestocksolution(200X)isenoughforone96-wellplate.Unusedstocksolutionshouldbealiquotedandstoredat<-20oC.Keepfromlightandavoidrepeatedfreeze-and-thawcycles.
1.2 Prepare0.3%β-mercaptoethanolassaybuffer:Add30µLofb-mercaptoethanol(ComponentF)to10mLofReactionBuffer(ComponentB),andmixwell.
Note:Additionalbufferisneededforpreparingaserialdilutionofβ-galactosidasesamples,whichisusedtogenerateastandardcurve.
1.3 Prepareassaymixture:Add25µLofresorufinβ-D-galactosidasestocksolution(fromStep1.1)into5mLofassaybuffer(fromStep1.2)tomakeassaymixture.
Note1:Thisβ-Galassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.
Note2:Onecandivideunusedβ-Galassaymixtureintosingleusealiquotsandstoredat-20oC.
2.Preparelysisbufferworkingsolution:
Makelysisbufferworkingsolutionbyadding5µLofβ-mercaptoethanol(ComponentF)to5mLofLysisBuffer(ComponentD)beforeuse.
Note:Alwaysadd0.1%β-mercaptoethanolintolysisbufferbeforelysingthecells.
3.Preparecellextractsfrommammaliancells:
3.1 TreatcellscontainingLacZgenewithtestcompoundsforadesiredperiodoftime.
3.2 Washthecellstwicewith1XPBS.Donotdislodgethecells.
3.3 Foradherentcells:Addlysisbufferworkingsolution(fromStep2)tothecultureplates.Recommendedvolumesforvariousplatesarelistedinthefollowingtable.
Typeofcultureplate | Volumeoflysisbufferworkingsolution(μL/well) |
96-wellplate | 50 |
24-wellplate | 250 |
12-wellplate | 500 |
6-wellplate | 1000 |
60mmplate | 2000 |
100mmplate | 4000 |
Fornon-adherentcells:Pelletthecellsintocentrifugetube,andadd50-2000µL(dependingonthesizeofthecellpellet)of1Xlysisbuffertothetube.
3.4 Incubatethecellswithcelllysisbuffer(fromStep3.3)atroomtemperaturefor10-15minutes,andgentlyswirltheplatesortubesseveraltimestoensurecompletelysis.
3.5 Proceedtotheβ-galactosidaseassayorfreezethesampleat-80oCtilluse.
Note1:Agoodlysiscanalsobeobtainedbyaquickfreeze-and-thawcycle(freeze1-2hoursat-20oCto‑80oCandthawatroomtemperature).
Note2:Alternatively,centrifugethecelllysisfor2-3minutestopellettheinsolublematerial,andruntestsonthesupernatant.
4.Runb-galactosidaseassay:
4.1 Thawthetubeorplateoflysedcellsatroomtemperatureifneeded.Performtheassaydirectlyonthe96-wellplateifthecellswereseededina96-wellplate.
4.2 Add50µL/wellofβ-galactosidasecontainingcellextracts(fromStep3.4)intoa96-wellsolidbackplate.
Note1:Ifnecessary,dilutethelysatein1XLysisBufferwhentransfectionefficiencyisveryhigh.Orreducethevolumeoflysisbufferwhentransfectionefficiencyislow.Ifthetransfectionisperformedina96-wellplate,orastablecelllinewasseededintoa96-wellplate,performtheassaydirectlyontheplate.
Note2:Forendogenousß-galactosidaseactivitycontrol,add50µLofcelllysatefromnon-transfectedcells.Forblankcontrol,add50µLoflysisbuffer.
4.3 Optional(ifastandardcurveisdesired):Prepareβ-galactosidase(E.Coli)standardsbyperforming1:3serialdilutionsin0.3%β-mercaptoethanolassaybuffer(fromStep1.2)togetapproximately100,30,10,3,1,0.3,0.1and0mU/mLβ-galactosidasestandards.Add50µL/wellofseriallydilutedb-galactosidasestandardsintoa96-wellsolidbackplate.
Note1:Adjustthestandardcurve(concentrationrange)tosuitthespecificexperimentalconditions,suchascelltype,number,transfectionefficiency,andsizeofthecultureplates.
Note2:Thedilutionsforthestandardcurvemustbepreparedfreshlyeachtimetheassayisperformed.
4.4 Add50µLofassaymixture(fromStep1.3)toeachwell.Incubatetheplateatroomtemperatureor37oCforapproximately10minutesto4hoursdependingonthecelltype.
4.5 Add50µLofStopBuffer(ComponentC)toeachwell.Thestopbuffercausesanincreaseinthefluorescenceintensityoftheproduct,inadditiontoterminatethereaction.
4.6 MonitortheabsorbanceincreasebymeasuringtheODratioatthewavelengthof580nmto460nm(Ab580/Ab460)usinganabsorbanceplatereader.
| References&Citations |  CitationExplorer |
BZLF1AttenuatesTransmissionofInflammatoryParacrineSenescenceinEpstein-BarrVirus-InfectedCellsbyDownregulatingTumorNecrosisFactorAlpha
Authors:XubingLong,YuqingLi,MengtianYang,LuHuang,WeijieGong,ErshengKuang
Journal:JournalofVirology(2016):7880--7893
