Lipid analysis：EXTRACTION OF LIPIDS FROM LIPOPHORIN相关交流-蚂蚁淘在线

EXTRACTIONOFLIPIDSFROMLIPOPHORIN

Sincelipidsarehydrophobic,theyarebettersolubleinorganicsolventsthaninwater.BecausethelipidsareburiedintoproteinparticlessimpleshakingoftheaqueousproteinsolutionwithanimmiscIBLesolventwillnotleadtoefficientextraction.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

UseonlyglassPipettesthroughoutthisexperiment.Alwaysmakeaflowchartoftheprocedurepriortocarryingouttheexperiment.

1.Retrievethelipophorinfromthefreeze-dryer,weighoutthelyophilizedpowderandputinasmall7mlglassvial.Closewithalidcontainingteflonlining.2.Toyourfreeze-driedlipophorin,add0.8mlwater.ShakevigorouslytosUSPendthelipoprotein.

3.Add10µlofinternallipidstandard(solutioncontains1mg/mlofallstandards)permgprotein(asweightedout).

4.Addchloroformandmethanol,mixinginthevortexandhomogenizingusingapasteurpipetteaftereachadditionsothatthefinalproportionsarenow

chloroform

methanol

water

0.8

5.Allowtostandfor10minatroomtemp.

6.Add1mlchloroformand1mlwater,mixingandhomogenizingasaboveaftereachaddition.Thefinalproportionsarenow

chloroform

methanol

water

1.8

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.

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Lipid&nbsp;analysis：EXTRACTION&nbsp;OF&nbsp;LIPIDS&nbsp;FROM&nbsp;LIPOPHORIN

Experimentalprotocol

Lipid analysis：EXTRACTION OF LIPIDS FROM LIPOPHORIN