EXTRACTIONOFLIPIDSFROMLIPOPHORIN Sincelipidsarehydrophobic,theyarebettersolubleinorganicsolventsthaninwater.BecausethelipidsareburiedintoproteinparticlessimpleshakingoftheaqueousproteinsolutionwithanimmiscIBLesolventwillnotleadtoefficientextraction. UseonlyglassPipettesthroughoutthisexperiment.Alwaysmakeaflowchartoftheprocedurepriortocarryingouttheexperiment. 1.Retrievethelipophorinfromthefreeze-dryer,weighoutthelyophilizedpowderandputinasmall7mlglassvial.Closewithalidcontainingteflonlining.2.Toyourfreeze-driedlipophorin,add0.8mlwater.ShakevigorouslytosUSPendthelipoprotein. 3.Add10µlofinternallipidstandard(solutioncontains1mg/mlofallstandards)permgprotein(asweightedout). 4.Addchloroformandmethanol,mixinginthevortexandhomogenizingusingapasteurpipetteaftereachadditionsothatthefinalproportionsarenow 5.Allowtostandfor10minatroomtemp. 6.Add1mlchloroformand1mlwater,mixingandhomogenizingasaboveaftereachaddition.Thefinalproportionsarenow 7.CentrifugeintheHermlecentrifuge(inB8220)for5minat4000rpmtoformtwolayers.(Uselargerubberadapterssothatthevialcanberemovedwithoutdisturbingthelayers).Thelower,chloroformlayercontainslipids.Theupperlayercontainswater,methanol,andwatersolublesalts. 8.Transferthelowerchloroformcontaininglayer(usingapasteurpipette)intocleanglassvials.Makesuretopreventcontaminationfromtheupperlayerortheinterphase.Ifthetransferredchloroformphasecontainswater(itmaylookcloudy),drythesolutionbyaddingaspatulafullofanhydroussodiumsulfate.Decantintoanewvial,andevaporatethesolvent(inthefumehood)inastreamofnitrogen. 9.Youshouldseesomeyellowsemi-liquidphaseatthebottomofthevial.Thisisyourlipidextract.UsetheTefloncoatedlids.Storeat-20°C.
Themethodofchoiceisaonephaseextraction,aspioneeredbyFolchandmodifiedbyBlighandDyer(1959).Water,methanol,andchloroformaremixedintheratio1:2:0.8.Undertheseconditions,allthreesolventsremainmixed,andthelipoproteinsareexposedtothesolventmolecules.Subsequently,waterandchloroformisaddedtoshifttherationto2:2:1.8,nowthepolarandnonpolarsolventsarenolongermiscible,andwegettwophases.Note:Chloroformisheavierthanwater.Thespeedandeaseofthismethodmakeitsuitableformultipleextractions,e.g.ofplasmasamples,providedthatasmallamountofofaqueouscontaminationcanbetolerated.Experimentalprotocol
chloroform methanol water chloroform methanol water
