Contributor:SupryaJayadevDate:Nov.10,1995Buffers:-2Xbuffer10ml0.5Mimidazol,pH6.60.21gLiCl1.25ml1MMgCl21.0ml0.1MEGTA,pH6.6-->Bringvolumeupto50mlwithdistilledwater.-dilutionbuffer1ml0.5Mimidazol,pH6.62.5ml20mMdiethylenetriaminepentaaceticacid(DTPA)-->Bringvolumeupto50mlwithdistilledwater.ReactionMixture:solution[stock]vol/tube#samplestotalvol.2Xbuffer-50µlX_______=_______DTT1M0.2µlX_______=_______DGKmembrane3.8µg/µl0.44µlX_______=_______dilutionbuffer-19.36µlX_______=_______total=_______1)Treatandharvestcells.2)ExtractlipidsviaabasicBligh-Dyerextraction:a)ResUSPendcellpelletin3mlchloroform/methanol(1:2)&vortex.b)Add0.8mlwater&vortex.c)Pelletcellulardebrisbyspinningat3,000rpm(2000xg)for5min.d)Add1mlchloroform&vortex.e)Add1mlwater&vortex.f)Discardupper,aqueousphase.3)Collect1.2-1.5mlofthelowerphase(totalvolumeoflowerphaseshouldconstitute2ml,buttopreventanyaqueouscontaminationonlyaportionisused).--->Alsoaliquotstandards:10,20,40,80&160pmolesofceramideanddiolein.4)Drydownlipidsusingextradrynitrogengas.5)Resuspendlipidsineachtubein20µlofb-OG/DOPG(7.5%:25mMmixedmicelle)andvortexvigorously.--->Haveablanktubecontainingb-OG/DOPGonly.6)Sonicatesamplesbrieflyandagainvortexvigorously.--->ThisstepisonlynecessaryifthelipidisnotreADIly"goingintosolution."7)Add70µlofreactionmixturetoeachsampleandvortexvigorously.8)Add10µlof2mM,4µCi[g-32P]ATPtoeachtubetoinitiatereaction.--->WhenpreparingthemixtureofhotandcoldATPassumethatthehotATPconstitutesinsignificantconcentrationsofATP.9)Allowreactiontoproceedfor30minutesatroomtemperature.10)Stopreactionwith3mlofchloroform/methanol(1:2)&vortex.11)Add0.7mlwatertoeachsample&vortex.--->Someprotocolsuse1%HClO4insteadofwateratthisstepandatstep13.12)Add1mlofchloroformtoeachsample&vortex.13)Add1mlwatertoeachsample&vortex.14)Spinsamples@3,000rpm(2000xg)for5minutes.15)Aspirateofftheupperphaseandcollect1.2-1.5mloflowerphase.NOTE:Theupperphasecontainsmostoftheradioactivity,sobeverycarefulwhenaspirating!16)Drydownsamples,resuspendinchloroformandspotonTLCplates(WhatmanSilicaGel60Aplates,catalog#:4866-821).17)DevelopplatesinTLCchambercontainingthefollowingsolventsystem:--->chloroform/acetone/methanol/aceticacid/water(10:4:3:2:1)18)Exposefilmovernight.19)Developfilm,scrapespots&countceramidephosphate,phosphatidicacidandlyso-phosphatidicacidspots.

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