Contributor:SupryaJayadevDate:Nov.10,19931)Incubatecellswith1µCi/mlof3H-galactosefor72hours.--->Iftreatmentisforanextendedperiodoftime:treatinserumfreemediacontaininglabeledgalactose.2)Washlabeloutandspincellsdown.3)Extractlipidsbywashing3timeswithchloroform-methanol(1:1).--->Use3mleachtime.4)Poolthesolventsfromtheextractionsanddrydownlipids.5)ResUSPendlipidsin2mlof0.1Nmethanolicsodiumhydroxide.6)Allowbasehydrolysistoproceedfor1hourat37°C.--->Basehydrolysisgetsridofanycontaminatingphospholipids.7)Neutralizebasebyadding267µlof0.75NHCl(anequimolarconcentrationofacidtobase).8)Add1mlofchloroformtobringthesolventratioto3:6:0.8ofchloroform-methanol-water.--->NOTE:Itisbestnottoextractatthispointbecausegangliosidescouldpotentiallypartitionintotheaqueousphaseandbelost.--->Thismixturecanbestoredinthefreezerforshortperiodsoftime.9)PrepareaslurryofDEAE-Sephadexinchloroform-methanol-water(30:60:8).--->Thisresinhasapositivechargeandwillthereforebindtheacidicglycosphingolipids.Thus,whenacombinationofneutralandacidiclipidsareadded,theacidiclipidswillstickandtheneutrallipidswillwashthrough.10)PackaBioraddispocolumnwith4mlofpackedgel,usechloroform-methanol-water(30:60:8)topackcolumn.11)Applysampletocolumn,washthesampletube2timeswith2mlofchloroform-methanol-water(3:6:0.8)andapplythataswell.12)Elutecolumnasfollows:a)30mlofchloroform-methanol-water(30:60:8)--->Thiswillcontinuetoelutetheneutralglycosphingolipids.b)30mlofchloroform-methanol-0.8MNaOH(30:60:8)--->Thisshouldelutetheacidicglycosphingolipids.13)Dryeachfractionintherotovap,resuspendlipidinaminimalvolumeofthesamesolventandtransfertoasmallscrew-cappedtube.14)Drydownsamplesagainand:a)Neutralglycosphingolipids:Resuspendinaminimalvolumeofchloroform-methanol(1:2),applytoTLCanddevelopinchloroform-methanol-water(65:25:4).b)Acidicglycosphingolipids:Resuspendin2mlofmethanol-1.6Msodiumacetate(1:1),desaltandapplysamplestoTLC.

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