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Labeled Sphingomyelin Synthesis

蚂蚁淘 /发布时间:1545758240 /浏览量:213

Contributor:SupryaJayadevDate:Mar.11,1993Synthesisreaction:1)MixpurifiedDMSMwithcyclohexylamineand14CH3Iataratioof1/1.1/1.3in5mlofmethanol.2)Allowreactiontoproceedatroomtemperatureforatleast18hoursinthedark.3)CheckcompletionofreactionbyrunningafewmicrolitersofthereactionmixonTLC(solventsystem=chloroform/methanol/ammoniumhydroxide@60:35:8).4)Oncereactioniscomplete,drythemixtureasmuchaspossIBLeusingnitrogengas.-->Shouldbeleftwithawhiteprecipitateinasmallvolumeofoily,yellowresidue.

Washingreactionproducts:5)Dissolvetheresiduein2mlofchloroformandvortex.6)Add1mlof5%Na2S2O3·5H2O(sodiumthiosulfate)andvortexvigorously.7)Spin@3,000rpmfor5minanddiscardupper,aqueousphaseinrADIoactivewaste.8)Washorganicphaseagainwithsodiumthiosulfate.9)Washorganicphasefromstep(8)twicewith2NHCl,discardupperphasesinradioactivewaste.10)WashtheacidifiedorganicphasetwicewithH2O,againdiscardupperphasesinradioactivewaste.11)DrydownwashedproductandresUSPendin2mlofchloroform.-->Ifaprecipitatepersists,alittlemethanolmaybeaddedtocompletelydissolvethematerial.

ColumnpurificationoflabeledSM:12)PackaBio-SilAcolumnasfollows:a)Packbaseofcolumnwithasmallportionofglasswool.b)Suspend20gramsof100-200meshBioSilAin100mlchloroform.c)Poursilica/chloroformmixtureintocolumnelutingchloroformfrombase.-Pressoutanyairbubblesintheglasswoolasthegelisinitiallybeingpoured.Whilepouringcolumnmatrix,stircontinuouslywithastirbarinordertopreventbubbleformationinthecolumn.d)Washcolumnthroughwith"200mlchloroform.-ALWAYSmaintainafluidlevelabovethepackingsilica...NEVERletcolumndryout!!13)Carefullylayerchloroformsolutionoverpackedcolumn.14)Allowsampletorunintocolumnwhilemaintainingasmalllevelofchloroformabovethepackedsilica.15)Sequentiallyelutewiththefollowingsolvents:a)150mlchloroformb)150mlacetonec)300mlacetone/methanol(9:1)d)50mlmethanole)100mlmethanolf)300mlmethanolg)300mlmethanol16)Drydownfractions(d)-(g)separatelyandre-suspendin4mloftoluene/methanol(1:1).17)Spot2µlofeachfractionandasmallportionofDMSM&SMstandardontoaTLCplate.18)RunTLCinchloroform/methanol/ammoniumhydroxide(60:35:8)anddevelopspotsusingiodinevaporthenpotassiumpermanganatespray.-->Fraction(d)&(e)shouldcontainunreactedDMSM,andfraction(f)shouldcontainthelabeledSM.19)StorelabeledSMin-20°Cfreezer.

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Labeled&nbsp;Sphingomyelin&nbsp;Synthesis

Labeled Sphingomyelin Synthesis