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1、Percolldensitygradientcentrifugation Outline PrinciplesPhysicalpropertiesStorageMakeandusedensityofPercollTipsProtocolInstrumentandoperationReferencesandapplicationsContrast differentialvelocitycentrifugation Principlesofdensitygradientcentrifugation Separationbydensity isopycniccentrifugation Separ
2、ationbysize ratezonalcentrifugation Percoll physicalproperties PercollisavailablefromGEHealthcare Compositionsilicasolwithnondialyzablepolyvinylpyrrolidone PVP coating 由聚乙烯吡咯烷酮包裹的硅胶颗粒 Density1 130 0 005g mlConductivity1 0mS cm 电导率 Osmolality 25mOsm kgH2O 渗透压 Viscosity10 5cPat20 C 粘度 pH9 0 0 5at20 CR
3、efractiveIndex1 3540 0 005at20 C 折射率 Percollisnon toxic 1 0 1 3g ml TheviscosityofPercollislowerinsalinesolutionsatphysiologicalionic StorageofPercoll Sterileandunopen 5yearsWhenopened Percollshouldbestoredbelow 8 IfPercollopenedundernon sterileconditions itcanbefrozenforupto6minthsat 18 Preformedgr
4、adientscanbestoredforweekswithoutachangeingradientshape providedthatthegradientissterileandisnotphysicallydisturbedIfstoredat 18 C gradientsformuponthawing necessitatingamixingofthecontentsofthebottlebeforeuse SterilizationofPercollsolutions 120 Cfor30minAbsenceofsaltsorsucrose Minimumcontactwithair
5、 narrow neckedbottle IfPercollformparticles theseparticlesmayberemovedbylowspeedcentrifugation Ifanysignificantevaporationoccursduringautoclaving thevolumeshouldbereplenishedwithsterilewatersothatthedensityisnotaffected HowtomakeandusegradientsofPercoll MakinganddilutingastocksolutionofPercoll Where
6、Vx volumeofdilutingmedium ml Vo volumeofundilutedPercoll ml o densityofPercoll 1 130 0 005g ml 10 densityof1 5MNaCl 1 058g mldensityof2 5Msucrose 1 316g ml i densityofSIPsolutionproduced g ml Thus forSIPinsaline i 1 123g ml forSIPinsucrose i 1 149g ml assuming o 1 130g ml Adding9parts v v ofPercollt
7、o1part v v of1 5MNaClor10 concentratedcellculturemediumisasimplewayofpreparingaStockIsotonicPercoll 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 HowtomakeandusegradientsofPercoll Dilutingstocksolutionstolowerdensities SolutionsofStockIsotonicPercoll SIP aredilutedtolowerdensit
8、iessimplybyadding0 15MNaCl ornormalstrengthcellculturemedium forcellwork orwith0 25Msucrosewhenworkingwithsubcellularparticlesorviruses TipsofmakingandusegradientsofPercoll Thecolloiddoesnotperceptiblydiffuseovertime resultingintheformationofverystablegradients Therefore bothdiscontinuousandcontinuo
9、usgradientscanbepreparedweeksinadvance givinggreatreproducibilityoverthecourseofanexperiment Measuretheweightofthesolution makesuretheweightofsolutionareallthesame 实验重复性的保证 1 Percollwasdiluted9 1 vol vol with1 5MNaCl 2 Put10mlofthePercollsolutioninto15 mlCorextubesandcentrifugedat19 240gavfor15minat
10、20 C swingingbucketrotor 3 Approximately2 109cells 200OD600 werepelleted resuspendedin1mlTrisbuffer overlaidontothepreformedgradient andcentrifugedat400gavfor60min Isolationofquiescentandnonquiescentcellsformyeaststationary phaseculture AllenC TheJournalofcellbiology 2006 Percollgradientpurification
11、ofspores StrainsweregrownovernightinYPDliquidmedium CellswerewashedtwicewithddH2OanddilutedtoafinalcelldensityofOD600 0 5 Then 10 lofequal volumemixedcellswerespottedonV8mediumandincubatedforsevendaysinthedarkatroomtemperature Theentirematingpatchwassuspendedin60 Percoll GEHealth inPBSwith0 1 Triton
12、X100 Aftercentrifugationat10 000Xgfor30minsinanSW41Tiultracentrifugerotor Beckman Coulter abandofsporesnearthebottomofthePercollgradientwasrecoveredwitha1 mltuberculinsyringeandtransferredintoanEppendorftube Thetotalsporeproductionwasdeterminedbymultiplyingthesporedensity measuredbyhemocytometer withthefinalvolume ChristinaM HullPLoSgenetics 2015 Instrumentandoperation Instrumentandoperation Instrumentandoperation Instrumentandoperation dataexport Referencesandapplications contrast
