bGalactosidase Activity Assay相关交流-蚂蚁淘在线

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
Calculatetheunitsofactivity^6.

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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bGalactosidase&nbsp;Activity&nbsp;Assay

bGalactosidase Activity Assay