Contributor:SupryaJayadevDate:Oct.20,1994Reagents:2NmethanolicNaOH2NHCl
Bligh&Dyerextraction1)Pelletapproximately1X107cells.2)ResUSPendpelletin3mlCHCl3:CH3OH(1:2)andvortexhard.3)Add0.8mlH2O,vortexhardandallowsolutiontositatroomtemperatureforatleast30minutes.4)Pelletcellulardebrisbyspinningat3,000rpm(inthetabletopcentrifuge)for5minutes.5)Transfersolventstoanewtubewithouttransferringanyofthepelleteddebris.6)Add1mlofCHCl3and1mlofH2Otoeachsample.-->UponadditionofCHCl3,abiphasicsolutionshouldresult.7)Vortexhardandspinsamplesat3,000rpm(inthetabletopcentrifuge)for5minutes.8)Removetheupperaqueous/methanolicphaseandtransferthelowerCHCl3phasetoanewtube.-->TheCHCl3phaseshouldconstitute2mltotalvolume.9)Drydownsolventunderextradrynitrogengas.10)Resuspendlipidsin1mlofCHCl3,aliquot800µloflipidsuspensionforbasehydrolysisand100µlforphosphatemeasurement.Basehydrolysis9)Bringlipidsupto1mlvolumeinCHCl3.10)Add100µlof2NmethanolicNaOHtoeachsample.-->Thefinalconcentrationofbaseis0.2N.11)Incubatesamplesat37°Cfor1hour.12)Neutralizebasebyadding100µlof2NHCltoeachsample.13)Add2mlCH3OHand600µlofwatertoeachsample,vortexhard.14)Allowsamplestositatroomtemperatureforatleast30minutes.15)Add1mlCHCl3tobreakphasesandvortexhard.16)Completetheextractionbyadditionof1mlofwater,vortexhard.17)Spinsamplesat3,000rpm(inthetabletopcentrifuge)for5minutes.18)AspirateofftheupperH2O/methanolicphaseandcollectthelower,CHCl3phase.19)Drythelipidsdownunderextradrynitrogengas,resuspendthemin75µlofCHCl3,aliquot10µlforphosphatemeasurementsandspot50µlonaTLCplate.20)Spotstandards(1-100nmolesSM)anddevelopplatesinCHCl3:CH3OH:Aceticacid:H2O(50:30:8:5).21)Visualizelipidsusingiodinevapor,mark,andscrapetheSMspotsintoa13x100screw-cappedtube.22)ExtracttheSMfromthesilicaasfollows:a)washwithCHCl3:CH3OH(2:1)2timesb)washwithCHCl3:CH3OH(1:2)onetime.23)QuantitatetheextractedSMviaastandardphosphateassay.
