DNA-MethyltransferaseAssay

ThisprotocolwaswrittenbyJean-PierreIssa,basedonAdamsetal.*Kam-WingJairhasmadesomeusefulshortcutsthatworkwellifyouarecareful.HereisKam-WingJair"sversionoftheassay.Thisassaycanbeusedtomeasureactivityinsmall,snapfrozenpiecesoftissue(e.g.,colonpolyps),orinculturedcells.

1.HomogenizeCells/Tissues

Forcellsinculture:Harvestlogphasegrowingcells(trypsinizationisOK),washtwiceinPBS,pelletbycentrifugation(2000rpm,5minintabletopcentrifuge,or3mininmicrofuge),resUSPendcarefullyandcompletelyinLysisBuffer(seesolutions),incubateatRT5minwithfrequentvortexing,andfreezeinETOH/dryicebath.Repeatfreeze/thawcycletwice(3timestotal)andstoreat-70°Cuntilreadytoquantitateproteins.Trytosuspend105cells/l5mlLB.Thenumberofcellsneednotbeexact,butyouneedaminimumof5x105cellsin75mltohaveaccurateresults,andifyouhavetoomanycellsinasmallvolume,theywillnotresuspendorlysewell.

Forfrozentissues:HomogenizewellinLB(200-300mlisappropriateforcolonbiopsiesandpolyps),usingatissuehomogenizerifpossIBLe,passthesolutionthrougha20gaugeneedlex2,followedbya25gaugeneedlex2,andfreezeinanETOH/dryicebath.Repeatfreeze/thawcyclex2(3timestotal),andstorelysateat-70°Cuntilreadytoquantitateproteins.

2.QuantitateProteinConcentration

Quantitateproteinsin15mlaliquotsoflysates(induplicate)byusingaBradfordassay(IhavebeenusingtheBioradkit),usingBSAasstandard.Don"tforgettoadd15mlLBtoeachBSAsampleandtotheblanktube.UsethesameLBbatchasthatusedtolysethecells.105cellsin15mlusuallyyields10-25mgprotein.Ifyouneedthegorydetails:Thawunknowns(oneatatime),aliquot15mlintotwo1.5mltubes,add200mlBioradstain(undiluted)and785ml0.15MNaclandmix.Preparestandardcurvebymixing2.5,5,10.15,25and30mgBSAwith15mlLB,200mlBioradstainand785ml0.15MNacl.AlsoprepareblanktubeswithLB,stainandNaC1butnoprotein.SetSpectrophotometeronOD595,zerousingblanks,measureODofstandardsandunknowns,andreadunknownconcentrationfromstandardcurve.Itisconvenienttoaliquot30mloflysateatthesametimeyoualiquotfortheproteinassay,andfreezethisaliquot,tobedilutedlater.

3.DiluteSamples

Afterquantitatingproteins,dilute30mlaliquotsbyaddingLB.Forculturedcells,Ihavebeendilutingto0.5mgproteinper15mlLB.Forfrozentissues,Ihavebeendilutingto5mgproteinper15ml.Thisassayislinearupto4mgproteinfromculturedcellsand10mgproteinfromwholetissue,butthelinearityislostatlowerconcentrationsifyouuseabadbatchoranoldbatchofSAM,anditisthereforebettertoworkwiththelowestconcentrationthatgivesinterpretableresults.Forthesamereason,itisbesttohaveallsamplesassayedatthesameconcentration,IfindoubtaboutyoursamplesorthequalityofSAM,determineactivityattwoconcentrationsordoaconcentrationvsactivitycuvepriortoassayingallsamples.

4.TheAssayProper

Mix15mllysatewith2mlpoly[d(I-C).d(I-C)1(Pharmacia,dilutedto0.25mg/mlusinglowTE),and3mlSAM(AmershamTRK581).Toavoidbubbles,pipetallsolutionsontothewallofthemicrofugetubes,andspin2-3stomixthesamples.

• Incubateat37°Cfortwohours.
• Add350mlStopSolution.
• Incubateat37°Cfor30min.(optional).
• Add350mlPCI-9,mixbyvortexing,spininmicrofuge30s,transferthesupernatanttoanewtubeandrepeatxl(2PCIextractionstotal).
• Add350mlchloroform,mixbyvortexing,spin30sandtransfersupernatanttoacleantube.
• Add700mlabsoluteETOH(keptat-20°C),mixbyinvertingtubestwo-threetimes,andplacein-70⁰Cfreezerfor15minor-20°Cfreezerfor30min,Theassaycanbeinterruptedatthispoint,andthesampleskeptat-20°Cuntilreadytoproceed.
• Vortex5-10s
• Spinincoldroommicrofugefor15min.
• Pouroffsupernatant.
• Add500ml70%ETOH.Vortex.
• Spinincoldroommicrofuge10min.
• Pouroffsupernatant.
• PipetoffresidualETOH.Alternatively,dryinspeedvacfor10min,butthepelletdoesnothavetobedrytoproceed.
• Resuspendthepelletin30ml0.3MNaOH,vortex5s,spinbrieflytobringsolutiontobottom.
• Incubateat37°Cfor45-60min.
• Spinbriefly.
• SpotsolutionontoGF/CWhatmanfilterpapers(2.4cm2fitsbestthecurrentmanifold).
• Dry5min.in80°Coven.
• Placefiltersonmanifoldwithsuctionoff.
• Pour1-2mlicecold5%TCAwithBSA(3mlof10mg/mlsolutionfor500mlTCA).
• Wait10sec.
• Turnonvacuum.Washfilterwith2-5mlTCA/BSA,followedby2-3ml70%ETOH.
• Dryin80°Covenfor5min.
• Placefiltersinscintillationvials.
• Add5mlEconofluor.
• Shakemanually5s.
• Countinliquidscintillationcounter(3Hchannel)
• Etvoila!

5.Troubleshooting

Anydeviationfromthisprotocolwillbesternlypunished!Butseriously,donotassumethatsomeofthesestepsaresuperfluous.Forexample,twoPCIextractionsreallyarenecessary,dryingoffiltersinovenisessential,etc.Ifyouthinkyoucanshorten/improvethisassay,pleasedo,butmakesureyoucheckyourmodificationagainstthisassay,checkingbothpositiveandnegativecontrols.Runallsamplesinduplicate.BecausethequalityofSAMvariestremendouslyfrombatchtobatch,runapositivecontrolwitheachassay.IfwereceiveanewSAMbatch,trytocompareoldvsnewbatchinthesameassay,usingthesamesample(thiswouldgreatlysimplifycorrectionfactors).Also,ifyouarethefirsttouseanewSAMbatchaliquotitinto75mlaliquotsbecauserepeatedfreeze/thawingofSAMacceleratesitsdegradation.

Finally,runanegativecontrolwitheachassay.Thisshouldbethesamesampleasthepositivecontrol,withSAM,butwithnodIdC.Ifyournegativecontrolsaretoohot:DidyouincludedIdCbymistake?Isthegridofthemanifoldcontaminated?(youcancheckbyusingablankfilter,washingwithTCA,dryingandcountinginEconofluor)Also,alongerNaOHincubationmightreducethisproblemsome.Butremember,thehighertheactivity,thehigherthenegativecontrolwillbebecauseofmethylationofendogenousDNA.Bythesametoken,ahighercellularproteinconcentrationwillalsoleadtoahighernegativecontrolforsimilarreasons(+proteinmethylation).

Ifyourpositivecontrolsaretoolow:Didyoudeviatefromprotocol?(e.g.,notdryingwilldefinitelylowercounts).Didyoulosepelletduring70%ETOHwash(yes,ithappenstothebestofus--andtheworst).AreyoudealingwithabadordegradedbatchofSAM?(sofar,onebatchwasbadbecauseitstayedinthefreezerforsixmonthsbeforebeingused,onebatchwasworthlessandreplacedfreeofchargebyAmersham,twobatcheswereaverageandonebatchwasgreat).Ifnoneoftheabove,maybethegodswereagainstyouonthatday--repeat!Onelastthing:Iusuallyrun24samplesatatime(i.e.,onenegative,omepositivecontrols,and11unknownsinduplicate).Twenty-fourwaschosenbecausethemicrofugewillnottakemoresamples,anddoingmorethanthatonasingledayis,Ibelieve,crazy.Ittakes30minutestosetupreactions,twohoursofincubation,onetotwohoursofstop/PCI,andtwotothreehourstofinishtheassay.

Goodluck.

6.Solutions

LysisBuffer

• Stock:728mlH₂O
• 5ml1MTrispH8.0
• 200ml0.5MEDTA
• 1ml1%NaAzide
• 10mlGlycerol
• 1mlTween80
• (StoreatRT)
• AddFresh:5ml0.2MDTTpermlLB,5mlPMSFpermlLB(PMSFstock:12mg/mlinIsopropranolol)

StopSolution

• Stock:50mlH₂O
• 10ml10%SDS
• 400ml0.5MEDTA
• 4gm4-Aminosalicylate(Nasalt,Sigma)
• 5mlButanol
• 12.5ml1MNaCl
• 8.3mlSalmontestisDNA(sonicated,stock:3mg/ml)
• H₂Oto100ml
• (Storeat4°C)
• AddFresh:ProteinaseK,50mlof20mg/mlstockpermlstopsolution

*Adaptedfrom:RLPAdamset.al.,JournalofBiochemicalandBiophysicalMethods,22:19-22,1991