Percoll 密度梯度离心教程.ppt

Percolldensitygradientcentrifugation,Outline,PrinciplesPhysicalpropertiesStorageMakeandusedensityofPercollTipsProtocolInstrumentandoperationReferencesandapplicationsContrast(differentialvelocitycentrifugation),Forbiologicalparticles,theidealgradientmediumhasbeendescribedasonehavingthefollowingcharacteristics:coversasufficientdensityrangeforisopycnicbandingofallbiologicalparticlesofinterestpossessesphysiologicalionicstrengthandpHisiso-osmoticthroughoutthegradienthaslowviscosityisnon-toxicwillnotpenetratebiologicalmembranesissuppliedsterileandisresterilizablewillformself-generatedgradientsbycentrifugationatmoderateg-forcesiscompatiblewithbiologicalmaterialsiseasilyremovedfrompurifiedmaterialsdoesnotaffectassayprocedureswillnotquenchradioactiveassays,Principlesofdensitygradientcentrifugation,Separationbydensity(isopycniccentrifugation),Separationbysize(ratezonalcentrifugation),Percollphysicalproperties,PercollisavailablefromGEHealthcare.Compositionsilicasolwithnondialyzablepolyvinylpyrrolidone(PVP)coating（由聚乙烯吡咯烷酮包裹的硅胶颗粒）Density1.1300.005g/mlConductivity1.0mS/cm（电导率）Osmolality25mOsm/kgH2O（渗透压）Viscosity105cPat20C（粘度）pH9.00.5at20CRefractiveIndex1.35400.005at20C（折射率）Percollisnon-toxic,1.01.3g/ml,TheviscosityofPercollislowerinsalinesolutionsatphysiologicalionic,StorageofPercoll,Sterileandunopen-5yearsWhenopened,Percollshouldbestoredbelow+8IfPercollopenedundernon-sterileconditions,itcanbefrozenforupto6minthsat-18Preformedgradientscanbestoredforweekswithoutachangeingradientshape,providedthatthegradientissterileandisnotphysicallydisturbedIfstoredat-18C,gradientsformuponthawing,necessitatingamixingofthecontentsofthebottlebeforeuse.,SterilizationofPercollsolutions,120Cfor30minAbsenceofsaltsorsucrose.Minimumcontactwithair(narrow-neckedbottle)IfPercollformparticles,theseparticlesmayberemovedbylowspeedcentrifugation.Ifanysignificantevaporationoccursduringautoclaving,thevolumeshouldbereplenishedwithsterilewatersothatthedensityisnotaffected.,HowtomakeandusegradientsofPercollMakinganddilutingastocksolutionofPercoll,WhereVx=volumeofdilutingmedium(ml)Vo=volumeofundilutedPercoll(ml)o=densityofPercoll(1.130+0.005g/ml*)10=densityof1.5MNaCl=1.058g/mldensityof2.5Msucrose=1.316g/mli=densityofSIPsolutionproduced(g/ml)Thus,forSIPinsaline,i=1.123g/ml,forSIPinsucrose,i=1.149g/ml,assumingo=1.130g/ml.,Adding9parts(v/v)ofPercollto1part(v/v)of1.5MNaClor10concentratedcellculturemediumisasimplewayofpreparingaStockIsotonicPercoll(SIP)solution.,“渗透活性物质的物质的量除以溶液的体积称为溶液的渗透浓度（osmolarity），单位为mol/L或mmol/L”,HowtomakeandusegradientsofPercollDilutingstocksolutionstolowerdensities,SolutionsofStockIsotonicPercoll(SIP)aredilutedtolowerdensitiessimplybyadding0.15MNaCl(ornormalstrengthcellculturemedium)forcellwork,orwith0.25Msucrosewhenworkingwithsubcellularparticlesorviruses.,TipsofmakingandusegradientsofPercoll,Thecolloiddoesnotperceptiblydiffuseovertime,resultingintheformationofverystablegradients.Therefore,bothdiscontinuousandcontinuousgradientscanbepreparedweeksinadvance,givinggreatreproducibilityoverthecourseofanexperiment.Measuretheweightofthesolution,makesuretheweightofsolutionareallthesame.,实验重复性的保证,1.Percollwasdiluted9:1(vol/vol)with1.5MNaCl,2.Put10mlofthePercollsolutioninto15-mlCorextubesandcentrifugedat19,240gavfor15minat20C.(swingingbucketrotor)3.Approximately2109cells(200OD600)werepelleted,resuspendedin1mlTrisbuffer,overlaidontothepreformedgradient,andcentrifugedat400gavfor60min.,Isolationofquiescentandnonquiescentcellsformyeaststationary-phaseculture,AllenC.TheJournalofcellbiology.2006,Percollgradientpurificationofspores,StrainsweregrownovernightinYPDliquidmedium.CellswerewashedtwicewithddH2OanddilutedtoafinalcelldensityofOD600=0.5.Then,10lofequal-volumemixedcellswerespottedonV8mediumandincubatedforsevendaysinthedarkatroomtemperature.Theentirematingpatchwassuspendedin60%Percoll(GEHealth)inPBSwith0.1%TritonX100.Aftercentrifugationat10,000Xgfor30minsinanSW41Tiultracentrifugerotor(Beckman-Coulter),abandofsporesnearthebottomofthePercollgradientwasrecoveredwitha1-mltuberculinsyringeandtransferredintoanEppendorftube.Thetotalsporeproductionwasdeterminedbymultiplyingthesporedensit