Contributor:SupryaJayadevDate:November4,1991Extraction:
1)Followingspin,savesupernatantforanalysis.Beextremelycarefulnottodisturbthepelletsinceitissomewhatdispersed.2)Immediatelyuponremovingsupernatantfromeachtube,add3mlsofchloroform-methanol,1:2,andvortexvigorously!!-->ItisnecessarytoaddtheC:Mimmediatelyinordertogetgooddispersionofthepellet.IftoomuchtimepassespriortoresUSPension,thepelletdoesnotdissolvewellandsonicationisnecessary.3)Add0.8mlofwaterandvortexvigorously.-->Stillhaveamonophaseanditmaybebesttoallowthismonophasetoequilibrateforalittlewhile.-->Thisisagoodpointtostopbystoringthesamplesinthe-200Cfreezer.4)Spinoutalllargedebrisbyspinningat3,000rpmfor5mins.5)Transferthesupernatanttoanewpyrexglasstubeandvortexagain.Donottransferanyofthepellet,evenifitisnecessarytosacrificesomeofthesample.6)Add1mlofchloroformtobreakphasesandvortexvigorously.7)Add1mlofwaterandagainvortexvigorously.8)Spinsamplesat3,000rpmfor5mins.tocompletelyseparatephases.9)Aspirateoffthetopphaseandtheinterface.-->Removeenoughsuchthatnoaqueousmaterialremainsattheminiscus.10)Thelowerphaseshouldconstitute~2ml.Transfer1.5-1.7mlofthistoanewpyrexglasstube.-->Thisisagoodpointtostopbystoringthetransferredsamplesinthe-200Cfreezer.11)Drydownsamplescompletelyandresuspendlipidsin60µlofchloroform.12)Use20µltospotalinearKTLCplateforTLCsystem#1,use20µltospotalinearKTLCplateforTLCsystem#2.TLC:
A)AllowTLCtankstoequilibrate1-2hrswithsolventpriortorunning.SYSTEM#1-->SeparatesPA,TXB2,HHT,HETE&AA.ethylacetate/2,2,4-trimethylpentane/aceticacid/H2Oataratioof90:50:20:100SYSTEM#2-->SeparatesDAG&AA.benzene/diethylether/ethanol/NH3ataratioof100:80:4:0.2B)FollowingTLCremoveplatesandallowtoevaporatecompletely.Thisshouldtakeafewhours.-->Ifanotherplateneedstoberuninthesametankthenallowtanktoequilibrateanotherhourfollowingremovalofthefirstplate.C)Sprayplatewithenhance,spotwithrADIoactiveink,wrapinsaranwrapandplaceinacassette(nointensifyingscreennecessary).-->Spotinkinlocationswhereitwillnotbedisturbed.Inotherwords,donotputspotsbelowthesampleapplicationlevels,sincetheplatewillbeheldupsidedowntoscrapespots.18)Exposefilmforaminimumof40hours(3daysismuchbetter)inthe-800Cfreezer.19)Allowenhancetocompletelyevaporate.-->Sincetheplateiswrappedinsaranwrapduringdeveloping,enhanceremainswetontheplate,andthismustbeevaporatedinordertoretainallofthesilicawhenscraping.20)Followingfilmdevelopment,markofflocationsofspots.21)Holdplateupsidedownandtiltedoverglassineweighpapersandscrapespots.-->Addscrapingsintoprefilledplasticscintillationvialstominimizelossofsilicaandsample.22)Vortexvialsandcountspots,countvialsagain24hourslater.
