Thisprocedureinvolvesuseofhumanplasma,apotentiallydangeroussourceofblood-bornedisease.Wearlong-cuffedgloves,andeye-protection. Dayone: -HumanPlasmaispurchasedfromtheRed-Cross.Itshouldbe3-4daysold,fromtesting.Theplasmabagisemptiedbycuttingthelongtubewithapairofsharpscissors(becarefulnottosplash).Totheplasma(300mL)isadded3mLof100mMEDTA(sterilefiltered)toafinalconcentrationof1mM.ProteaseInhibitorsmayalsobeaddedaspreservative. -Plasma(~300mL)isspunat12C@20,000RPMfor20mininaTI-70Rotor.Thetubesareremovedandthechylomicronsarediscarded.Iftheplasmadonorfasted(asinstructed)thereshouldbelittlecyhlomicronsintheupperfraction.Discardtheupperwhite,chylomicronsandtransferthesamplestonewtubes. -Plasma(~250ml)isspunat12C@52,000RPMfor24hinTI-70-Rotor.~25mLpertube.Tubesusinganinvertedplugareused,withared,aluminumscrew-cap.TubesaresealedandplacedinUltraCentrifuge. DayTwo: -Mixthelowerlayer,leavingthegreenish-pelletintact.Thepelletcontainsglycogenandfibrinogen,andsomeLDL,butnotenoughtoworryabout.Tubesmaybeinvertedandsqueezedtoremovethecontents.Discardpellet,andtubes. -AdjustthedensityoftheLDL-Plasmato1.06usingPotassiumBromide(KBr).IfdensityisnotadjustedLDLwillnotspindown.CalibratePipettetoensureaccuracy,usingdH2O. -Add.075gKBr/mLPlasma. -Checkdensityweighing1mLofsolution.Ifnothighenough,addKBr. -Spinat12C@40,000RPMfor48hinTI-70Rotor. -Stoptherotor,withoutusingthebrake. -CollecttheLDL(uppermostfraction)takingcarenottomixthelayersorfractions. -TheLDLshouldbekeptundernitrogen,darkandat4Cuntiluse.(Thispreparationaccordingtoothersourcesisgoodforupto2weeks.)
