Centrifugation文档格式.docx

Centrifugation文档格式.docx

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Thefirstcommercialultracentrifugewasproducedin1940bySPINCO（notBeckmanCoulter）.

Improvementsleadingtomodernultracentrifuges:

Highspeeddrivesystems–themotor.

Methodstoreducefrictiongeneratedathighspeeds–developmentofvacuumsystems.Vacuumsystemsalsoenabledthemaintenanceofconstanttemperature.

Improvementsinrotormaterialtowithstandhighcentrifugalforces.Svedberg’sgroupusedhightensilesteel.Nowaluminumalloys,titaniumareused.

Improvementsinmetjodstoexaminetheparticlesbeingcentrifuged–betteropticalmethods.

Alsoimportantwasthedevelopmentoftechniquessuchaselectromicroscopywhichenabledtheearlyworkerstoseetheorganellesthattheywereseparatingwiththeultracentrifuge–abletoformulatestructure-functionrelationships.

Twotypesofultracentrifugesdeveloped:

analyticalandpreparative.

Analytical

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Usessmallsamplesize（lessthan1ml）

Builtinopticalsystemtoanalyzeprogressofmoleculesduringcentrifugation

Usesrelativelypuresample

UsedtopreciselydeterminesedimentationcoefficientandMWofmolecules

BeckmanModelEisanexampleofcentrifugeusedforthesepurposes.

Preparative

Largersamplesizecanbeused

Noopticalread-out–collectfractionsandanalyzethemaftertherun

Lasspuresamplecanbeused

CanbeusedtoestimatesedimentationcoefficientandMW

Generallyusedtoseparateorganellesandmolecules.Mostcentrifugationworkdoneusingpreparativeultracentrifuge

Severalmodelsavailable,includingL5-65andL5-75usedforpreparativepurposes.

SeparationMethodsusedinpreparativeultracentrifugation

Differentialcentrifugation–pelleting

Startwithauniformmixtureofsample–moleculesororganellesinbuffer.

Centrifugetoobtainpelletofheaviermolecules,andsupernatant

Becauseallsizedparticlesareevenlydistributedinthebeginning,thepelletwillcontainprimarilylargesizedparticles,butwillalsocontainsomeoftheothersizedparticles.Canre-suspendthepelletandre-centrifuge（2-3times）togetfairlypureprepofheaviestparticles,but100%separationisnotpossiblebythismethod.

Cantakesupernatantandre-centrifugetopelletthenextlargestparticlesuccessivecentrifugationatincreasinglyhighercentrifugalforcers（gforces）canpartiallypurifythedifferentsizedparticles.Toobtaingoodseparation,sizeanddensityoftheparticlesmustdifferbyseveralordersofmagnitude.

Commonlyusedforseparationoforganelles.

Usuallyuseafixedanglerotor.

Densitygradientcentrifugation:

Ratezonal（alsotermedsedimentationvelocity,zonecentrifugation）

Useacontinuosdensitygradientofsolventsuchassucrose.Desnsityincreasestowardthebottomoftube.Sampleislayeredontop.Centrifugeuntilmoleculesformdiscretebandsdependingupontheirsedimentationvalues.Generallystoptherunbeforeanyofthemoleculesreachesthebottom.100%separationispossiblewithasmallsample.

Separatesmoleculesbasedonsize（MW）.

Usedtoseparatevarioustypesofmacromolecules–mixtureofproteins;

differenttypesofRNA;

separateDNAfromRNA,orproteins;

ribosomalsubunits;

polyribosomes.

Swingingbucketalwaysrotorused.

Isopycniccentrifugation（alsotermedsedimentationequilibriumcentrifugation）

Mixgradientmaterial（suchascesiumchloride-CsCl）withsamplemolecules.

Duringthecentrifugation,theCsClgeneratesagradient（“self-generatinggradient”）,andthemoleculesmovetothepositioninthegadientwheretheirdensityisthesameasthegradientmaterial.Isopycnicmeans“samedensity,”sothemoleculesmovetotheir“isopycnicposition.”

Inordertogenerateagradient,youselectaCsClconcentrationthat,whenredistributedduringcentrifugationtoformagradient,willgiveyouarangeofdensitiesthaincludesthedensitiesofallthemoleculesyouwanttoseparate.CsClandothersaltsofalkalimetals（Cssulfate）areusedbecauseyoucangeneratehighdensitieswiththesematerials.

Requireslongcentrifugationtimetoformagradient（36-48hrs）.Canreducethetimeneededtoformagradient（byatleat1/2）bypreparingstep（discontinous）gradientsbeforecentrifugation.

Separatesmoleculesbasedontheirdensity–mayhavethesamesize.

OftenusedtoseparatevarioustypesofDNA–circularvslinear,doublestrandedvssinglestranded,DNAfromRNA（RNApellets）,highlyrepetitiveDNA（satelliteDNA）fromotherDnainthecell,canbeusedforseparationoflipoproteinsandcellorganelles.

SemiconservativereplicationofDNAwasdemonstratedusingisopycniccentrifugation.

Canuseswingingbucketorfixedanglerotor.

TheoryofCentrifugation

Sedimentationofamoleculesisinfluencedby…

Propertiesofthemolecules（size,shape,density）

Propertiesofthesolvent,orgradientmaterial（density,viscosity,temperature）

Interactionsbetweenthesolutemoleculesandthesolventgradientmaterial

Astherotorspinsinacentrifuge,centrifugalforceisappliedtoeachmoleculeinthesample:

Centrifugalforce=Mω2r

WhereM-mass（particleweight,ormolecularweight）;

w（omega）–angularvelocity（radieas/sec）;

r–distancefromtheaxisofrotation.

Thisequationsaysthatthelargerthemolecule,orthefasterthecentrifugation,orthelongertheaxisofrotation,thegreaterthecentrifugalforceandtherateofsedimentation.

Twoforcesacttocounteractthecentrifugalforce–thetwocounteractingforcesarethebuoyantforce（ordisplacementforce）andthefrictionalforce.

Bouyantforce=Mω2rVρ

WhereV–partialspecificvolumeofthesolute9molecules）-thevolumedisplacedbythemolecule.Definitions:

themlofsolutionvolumeincreasecausedbyadditionof1gofsolute;

volumeoccupiedwhen1gofsoluteisaddedtoalargevolumeofsolution.

ρ（rho）–densityofthesolvent（g/ml）.

Thedissolvedorsuspendedparticles（molecules）alsogeneratefrictionastheymigratethroughthesolution:

Frictionalforce=f（v）=f（dr/dt）

F–frictionalcoefficientuniquetothemoleculesinquestion.

dr/dt–rateofsedimentationexpressedasachangeintheaxisofrotationwithtime

Forasphericalmolecule,f=6πηrm

η–viscosityofthemediuminpoises（g/cmsec）

rm–radiusofparticle（molecule）

Sof,thefrictionalcoefficient,dependsuponthesizeandshapeofthemolecule,andontheviscosityofthegradientmaterial.

Asedimentingmoleculemovesfasterandfasterinacentrifugalfielduntilthecentrifugalforceequalstheconteractingbuoyantandfrictionalforces:

Centrifugalforce=buoyantforce+frictionalforce

Thisoccursbecausethefrictionalforceincreaseswithincreasedrateofsedimentationwhereascentrifugalforceandbuoyantforceareconstantforaparticularmoleculeandrotorspeed.Inpractice,thisbalancingofforcesoccursquicklywiththeresultthatamoleculessedimentsataconstantrate（dr/dt）.

Ifwesubstitutetheaboveequationsintothisrelationship…

Mω2r=Mω2rVρ+f（dr/dt）

Byrearranging…

Mω2r-Mω2rVρ=f（dr/dt）

M（1–Vρ）ω2r=f（dr/dt）

M=（f/1–Vρ）[（dr/dt）/ω2r]

Sedimentationcoefficient（s）

Anewtermthesedimentationcoefficient（s）describesthevelocityattainedbyaparticle（molecule）foragivenangularacceleration:

S=v/ω2r,wherevisvelocity,ors=（dr/dt）/ω2r

dr/dtisthechangeinaxisofrotationovertime.

Ifwesubstitute“s”intheaboveequation:

M=（f）（s）/1-Vρ

Or

S=M（1-Vρ）/f

Thefrictionalcoefficientf,canbeevaluatedthroughanexperimentallydetermineddiffusionconstant,D,where

D=RT/f

f=RT/C

R=gasconstant;

T=absolutetemperature.

Substitutingthetheaboveequation..

M=RTs/D（1-Vρ）

s=M（1-Vρ）D/RT

ThesearetheequationsthatareusedtoconvertM（molecularweight）ofamoleculeorparticletos,itssedimentationcoefficient（orviceversa）.

Sedimentationcoefficients（s）forbiologicalmoleculesfallbetween1and500x10-13seconds.Weavoidtheunitof10-13secondsbydefiningoneSvedbergunit,orSvedbers（Sas1x10-13sec.Ifwemeasurethesedimentationcoefficientofamoleculeas10-12sec,or