b-GalactosidaseActivityAssay--MarianPrice-Carter,9/7/00Day1:Startovernightculturesinassaymedium.Negativecontrol:cellslackingb-galactosidase,suchasLT2;positivecontrol:cellswithhighenzymeactivity.Day2:Dilutecells1/100infreshmedium,growtomid-log.1Preparesolutions:Zbuffer,phosphatebuffer,ONPG2.PreparationofCellsIncubatecultures20"onicetostopgrowthandwash:- Pelletatleast2mLofcellsat4Cbycentrifuging10"at6,000rpminaSorvalSS34rotor.
- Pouroffthesupernatant.
- ResUSPendthecellpelletinthesamevolumeofchilledZbuffer.
- MeasuretheOD600oftheresuspendedcells(blankagainstZbuffer)
DilutecellsinZbufferto1mL(mosteasilydonewithapippeter).Formostactivities,0.5mLcells+0.5mLZbufferwillproduceadesirableamountofyellowcolorin1-2hours.Forhigherlevels(>500Millerunits),try0.1mLcells+0.9mLZbuffer.PermeABIlizethedilutedcellsbyadding100µlchloroformand50µl0.1%SDS(sodiumdodecylsulfate,sodiumlaurelsulfate).Chloroformiseasiertopippeteiftheairinthepippetetipissaturatedbydrawingupandreleasingchloroformseveraltimes.Vortex;equilibratethetubes5"ina28Cwaterbath.AssayStartreactionbyadding0.2mLsubstrate,o-nitrophenyl-b-D-galactoside(ONPG;4mg/mL)- Vortex-Recordthetimeofadditionpreciselywithtimerorstopwatch.
- Incubatethecellsat28C.
- Stopthereactionaftersufficientyellowcolorhasdeveloped3byadding0.5mL1MNa2CO34.
- Vortex.-Notetimeofadditionprecisely.
- Transfer1mLtoanEppendorftube,spin5"atmaximumtoremovedebrisandchloroform.
- Recordtheopticaldensityat420nmandat550nmforeachtube.5
- Calculatetheunitsofactivity6.
ThisisbasicallytheassaydescribedbyJ.H.Millerin"ExperimentsinMolecularGenetics"1972ColdSpringHarborLaboratoriespages352-355,withanextrastepadded.Intheassaydescribedhere,thecellsarepelletedandresuspendedinassaybuffer(Zbuffer)toeliminateerrorduetotheeffectsofdifferentcarbonsourcesinthegrowthmediumontheb-galactosidaseenzymeactivity.b-Galactosidaseisabletohydrolyze(cleave)b-D-galactosides.Thisenzymefacilitatesgrowthoncarbonsourceslikelactosebycleavingitintoamoleculeofglucoseandamoleculeofgalactosewhichthecellscancatabolizeandgrowon.Intheassaydescribedabove,thesubstrateo-nitrophenyl-b-D-galactopyraniside(ONPG)isusedinplaceoflactose.Whentheb-galactosidasecleavesONPG,o-nitrophenolisreleased.Thiscompoundhasayellowcolor,andabsorbs420nmlight.Tomeasureb-galactosidaseactivitytheaccumulationofyellowcolor(increase420nmabsorbance)/minuteismonitored.Footnotes1InSalmonella(whichisnaturallyb-galactosidaseminus)thisassayisusedtomonitortranscriptionfrominsertionelements(thatencodetheb-galactosidaseenzyme)thathaveinsertedintodifferentgenes.Theassayisusuallyperformedoncellsinthemid-logphaseofgrowth.Onrichcarbonsourceslikeglucose,theOD600ofacultureofwild-typeSalmonellainmid-logphaserangesfrom0.28-0.7.Onpoorercarbonsourcesorinstrainsthathavemutationsingenesthatareimportantforgrowth,theOD600atmid-logphasemaybelower,sincethecellsmayenterstationaryphaseatalowerdensity.Therefore,beforedoingtheassay,itisimportanttofollowthegrowthofthestrainofinterestineachtypeofmediumthatwillbeused,plotagrowthcurve,anddeterminewhenthecellsareinmid-logphaseinthatparticularmedium.2Solutionsforb-galactosidaseassaysZbuffer,per50mL:- O.80gNa2HPO4.7H2O(0.06M)
- 0.28gNaH2PO4.H2O(0.04M)
- 0.5mL1MKCl(0.01M)
- 0.05mL1MMgSO4(0.001M)
- 0.135mLb-mercaptoethanol(BME)(0.05M)
- bringtoapproximately40mLwithH2O,dissolveallthesalts
- adjustthepHto7.0
- useagraduatedcylindertobringthebufferto50mL
- storeat4C.
Note:BMEisaddedtothereactionbuffertostabilizetheb-galactosidaseenzyme.TheimportantpartofBMEisareactivethiol(SHgroup).Thiolsreactwithoxygenintheairandoxidize(inactivate)overtime.Therefore,trynottomakemuchmoreZbufferthanyouwilluseinafewdays.Storetheunusedportionat4C.ONPGshouldbedissolvedfresheachday.Dissolve1.5Xasmuchasyouthinkyouwillneed,becauseyoumayhavetorepeatoneormoreoftheassaysi.e.foradifferentamountoftimeorwithadifferentcelldilution.DissolvetheONPGtoafinalconcentrationof4mg/mLin0.1MphosphatebufferpH7.0.Phosphatebuffer,per100mL:- 1.61gNa2HPO4.7H2O(0.06M)
- 0.55gNaH2PO4.H2O(0.04M)
- adjustthepHto7.0
- phosphatebufferisstableatroomtemperatureanddoesnotneedtobemadefresheachtime.
3Whatissufficientyellowcolor?Togetthemostaccuratemeasureofactivity,theabsorbanceat420nm(A420)shouldrangefrom0.6to0.9.ReADIngsaslowas0.1andashighas1.2areacceptable.Tubesthathavebecomeasyellowasatubeof(unused)LBbrothwillprobablybesufficientlyyellow.Ifthereadingistoolow,trytheassayagainwithmorecellsorlongerincubationtime.Whentheelementhasinsertedintoagenethatisnotexpressedmuch,itwillprobablytakehourstodevelopenoughyellowcolor.Ifyournegativecontrolstartstoturnyellow(afterseveralormorehours)itmeansthatthesubstrateisbeginningtoauto-hydrolyze.Theassaycanbeleftovernight.Theauto-hydrolysisisthenaccountedforbysubtractingtheA420andA500ofthenegativecontrolfromthatofthetestsbeforedoinganyfurthercalculations.Ifthereadingistoohigh,trytheassayagainwithfewercells.Aimtostopthereactionafter15minutes.Forexample,ifinyourfirstattempt,youadded0.5mLofcells+0.5mLofZbuffer,anditwastooyellowafter5minutes,tryadding0.1mLcells+0.9mLofZbuffer.Watchthetubecarefully.Someculturesmayhavetobedilutedevenfurther!4Addingthe1MNa2CO3stopsthereactionbyraisingthepHofthesolutionto11.AtthispHtheenzymeisnotactive.5Thereadingat420nmisacombinationofabsorbancebyo-nitrophenolandlightscatteringbycelldebris.Theabsorbanceat550correctsforlightscattering.Thereisnoabsorbancefromo-nitrophenolatthiswavelength.Thelightscatteringat420nmisproportionaltothatat550nm:lightscatteringat420nm=1.75xOD550
6Usethefollowingequationtocalculateunitsofenzymeactivity:MillerUnits=1000x[(OD420-1.75xOD550)]/(TxVxOD600)- OD420andOD550arereadfromthereactionmixture.
- OD600reflectscelldensityinthewashedcellsuspension.
- T=timeofthereactioninminutes.
- V=volumeofcultureusedintheassayinmLs.
TheunitsgivethechangeinA420/min/mLofcells/OD600 Typicalvalues:afullyinducedlac+operon(+IPTG)=1500unitsanuniducedlac+operon(noIPTG)=1.5-3units
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