RBC膜提取方法

Page1of27/12/06UniversityofCalifornia,SanDiegoCOSMOS2006,Cluster7BioengineeringandModelingoftheRedBloodCellMembraneTopic#1:Bioengineering,theAmazingRedBloodCellsLab4:Isolationofredbloodcellghostmembranes.Date:Mon,7/17/06Time:1:35–3:35pmInstructors:WeijuanYao&TerrellGreenA)Objectives:Isolatetheghostmembranefrommouseredbloodcells.B)MaterialsRequired:Mouseblood(200μl).WashBuffer,pH7.5–1L150mMNaCl5mMSodiumPhosphate:4ml–1MSodiumPhosphateDibasic1ml–1MSodiumPhosphateMonobasic20mgPMSF(Phenylmethylsulphonylfluoride)HypotonicLysisBuffer,pH7.5–1L7.5mMSodiumPhosphate6ml–1MSodiumPhosphateDidasic1.5ml-1MSodiumPhosphateMonobasic1mMEDTA20mgPMSF2mgPepstatinMicrocentrifugetubesPipettes(1000and200μl)DisposabletipsPipettorTransferpipettes(10ml)SuperspeedcentrifugemachineSuperspeedcentrifugetubesMagneticstirbarStirringplatesWeticeIcebucketC)PPErequired:LabcoatSafetyglasses/googlesGlovesClosedtoeshoesD)Background:Whentheredbloodcellsaresubjectedinthemediumwithlowersaltconcentrationthantheirinterior,waterwillenterintothecells.CellswillswellandthenPage2of27/12/06UniversityofCalifornia,SanDiegoCOSMOS2006,Cluster7BioengineeringandModelingoftheRedBloodCellMembraneburst,causingthehemoglobinsandothercytosolicproteinsrelease.Theremainedemptymembranesarecalledghostmembrane.Ghostmembranescanbecollectedbyhighspeedofcentrifugation.Theisolatedghostmembraneswillbeusedfortheproteinanalysis.E)Description/Protocol:1)Take200µlofbloodandpackRBCsbycentrifugationat1400xg,3min,4C.2)Aspirateplasmaandbuffycoat.EstimatethevolumeofpackedRBCs.3)WashRBCsbyresuspendingin4volumesofWashBuffer,andcentrifugeat1400xg,3min,4C.4)Aspiratesupernatantandrepeatwashstep3)once.5)EstimatethevolumeofpackedRBCs.Need~40volumeofHypotonicLysisBuffer.6)TransferHypotonicLysisBuffertoacentrifugetubewithstirbarincubatedinweticebath.7)LysebyaddingRBCsdropwisetocontinuouslystirringHypotonicLysisBuffer.OnceallpackedRBCsareadded,continuestirringfor2-3min.8)Centrifugeat22,000xg,20min,4C.9)CarefullyaspiratesupernatantliquidandWBCbutton,leavinglooselypackedghostmembranepellet.10)Washghostmembranepellettwicewith10mlHypotonicLysisBuffer.11)Finalpelletiswhitewithpinkcoatandsupernatantliquidiscolorless.Maximumyieldisapproximately60%ofpackedRBCsvolume.12)Takeoutthesupernatantasmuchaspossiblewithoutdisturbingthepellet.13)Resuspendthepelletintheremainedliquidandaliquotsamplesof50µlofghostmembraneat-80C.F)ExpectedResults:Eachgroupshouldseethelooselypackedghostmembrane.G)References:Dodge,J.T.,Mitchell,C.,Hanahan,D.J.,1963.Thepreparationandchemicalcharacteristicsofhemoglobinfreeghostsofhumanerythrocytes.Arch.Biochem.Biophys.100,119-130.H)Assignment:1.WhydoweusePMSFandpepstatininthewashbufferandhypotoniclysisbuffer?2.Whatisbuffycoat?Whywewanttogetridofit?