| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 490/514 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy ReporterGeneEnzymes |
| Related | TagEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareFDGworkingsolutionfor1plate:
1.1 Thawallthecomponentsatroomtemperaturebeforeuse.
1.2 MakeFDGstocksolution:Add125µLofDMSO(ComponentE)intothevialofFDG(ComponentA).
Note:25µLofFDGisenoughfor1plate.Un-usedFDGstocksolutionshouldbealiquotedandstoredat<-20oC.Keepfromlightandavoidrepeatedfreeze-and-thawcycles.
1.3 Make0.3%β-mercaptoethanolassaybuffer:Add30µLofb-mercaptoethanol(ComponentF)to10mLofReactionBuffer(ComponentB),andmixwell.
Note:Additionalbufferisneededforpreparingenzymedilutionbuffer,whichisusedtogenerateastandardcurve.
1.4 MakeFDGworkingsolution:Add25µLofFDGstocksolution(fromStep1.2)into5mLof0.3%β-mercaptoethanolassaybuffer(fromStep1.3).
Note1:DOnotkeepFDGsolutionsatroomtemperatureforanextendedperiodoftimeasspontaneoushydrolysiswilloccur.
Note2:Un-usedFDGsolutionscanbealiquotedandstoredat<-20oCformorethanonemonth.Keepfromlightandavoidrepeatedfreeze-and-thawcycles.
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 ProceedtotheFDGassayorfreezethesampleat-80oCtilluse.
Note1:Agoodlysiscanalsobeobtainedbyaquickfreeze-and-thawcycle(freeze1-2hoursat-20oCto‑80oCandthawatroomtemperature).
Note2:Alternatively,centrifugethecelllysisfor2-3minutestopellettheinsolublematerial,andthenassaythesupernatant.
4.Runb-galactosidaseassay:
4.1 Thawthetubeorplateoflysedcellsatroomtemperatureifneeded.Performtheassaydirectlyonthe96-wellplateifthecellswereseededina96-wellplate.
4.2 Add50µLofcellextracts(fromStep3.4)intoeachwellofthe96-wellplate.Savesomecontrolwellsforthestandardcurveifastandardcurveisdesired.
4.3 Optional(ifastandardcurveisdesired):Prepareaserialdilutionofβ-galactosidase(E.Coli)standardswith0.3%b-mercaptoethanolassaybuffer(fromStep1.3).Transfer50µLaliquotofeachpointonthestandardcurvetothecontrolwellsoftheplate.Thehighestrecommendedamountofb-galactosidaseis200mU(200-400ng).2Xserialdilutionofstandardcurveconsistingof8pointsisrecommended.Adilutionprocedureexampleisshowninthefollowingtable.
β-galStandard(mU) | AssayBufferVolume | β-galStandardVolume |
200 | 990µL | 10µLof20unitsβ-gal |
100 | 200µL | 200µLof200mUß-gal |
50 | 200µL | 200µLof100mUß-gal |
25 | 200µL | 200µLof50mUß-gal |
12.5 | 200µL | 200µLof25mUß-gal |
6.25 | 200µL | 200µLof12.5mUß-gal |
3.125 | 200µL | 200µLof6.25mUß-gal |
1.562 | 200µL | 200µLof3.125mUß-gal |
Note1:Adjustthestandardcurvetosuitthespecificexperimentalconditions,suchascelltype,number,transfectioneffeciency,andsizeofthecultureplates.
Note2:Thedilutionsforthestandardcurvemustbepreparedfreshlyeachtimetheassayisperformed.
4.4 Add50µLofeachsample/well.
Note1:Ifnecessary,dilutethelysatein1XLysisBufferwhentransfectionefficiencyisveryhigh.Orreducethevolumeoflysisbufferwhentransfectionefficiencyislow.Ifthetransfectionisperformedina96-wellplate,orastablecelllinewasseededintoa96-wellplate,performtheassaydirectlyontheplate.
Note2:Forendogenousß-galactosidaseactivitycontrol,add50µLofcelllysatefromnon-transfectedcells.Forblankcontrol,add50µLof1Xlysisbuffer.
4.5 Add50µLofFDGworkingsolution(fromStep1.4)toeachwell.Incubatetheplateatroomtemperatureor37oCforapproximately10minto4hrdependingonthecelltype.
4.6 Add50µLofStopBuffer(ComponentC)toeachwell.Thestopbuffercausesanincreaseinthefluorescenceintensityoftheproduct,inadditiontoterminatethereaction.
4.7 MeasurethefluorescenceintensityofthesolutionineachwellwithafluorescencemicroplatereaderatEx/Em=490/525nm.
4.8 Quantifyß-galactosidaseexpressionbasedonalinearstandardcurve.
| References&Citations |  CitationExplorer |
BZLF1AttenuatesTransmissionofInflammatoryParacrineSenescenceinEpstein-BarrVirus-InfectedCellsbyDownregulatingTumorNecrosisFactorAlpha
Authors:XubingLong,YuqingLi,MengtianYang,LuHuang,WeijieGong,ErshengKuang
Journal:JournalofVirology(2016):7880--7893
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