離心機參數(shù)計算

離心機參數(shù)計算第1頁，課件共25頁，創(chuàng)作于2023年2月CentrifugerotorsFixed-angleaxisofrotationAtrestSwinging-bucketgSpinningg第2頁，課件共25頁，創(chuàng)作于2023年2月GeometryofrotorsbcrmaxravrminrmaxravrminSedimentationpathlengthaxisofrotationarmaxravrmin第3頁，課件共25頁，創(chuàng)作于2023年2月k’-factorofrotorsThek’-factorisameasureofthetimetakenforaparticletosedimentthroughasucrosegradientThemostefficientrotorswhichoperateatahighRCFandhavealowsedimentationpathlengththereforehavethelowestk’-factorsThecentrifugationtimes(t)andk’-factorsfortwodifferentrotors(1and2)arerelatedby:第4頁，課件共25頁，創(chuàng)作于2023年2月CalculationofRCFandQRCF=RelativeCentrifugalForce(g-force)Q=rpm;r=radiusincm第5頁，課件共25頁，創(chuàng)作于2023年2月RCFinswinging-bucketandfixed-anglerotorsat40,000rpmBeckmanSW41swinging-bucket(13ml)gmin=119,850g;gav=196,770g;gmax=273,690gBeckman70.1Tifixed-anglerotor(13ml)gmin=72,450g;gav=109,120g;gmax=146,680g第6頁，課件共25頁，創(chuàng)作于2023年2月Velocityofsedimentationofaparticlev=velocityofsedimentationd=diameterofparticle

p=densityofparticle

l=densityofliquid

=viscosityofliquidg=centrifugalforce第7頁，課件共25頁，創(chuàng)作于2023年2月DifferentialcentrifugationDensityofliquidisuniformDensityofliquid<<DensityofparticlesViscosityoftheliquidislowConsequence: Rateofparticlesedimentationdependsmainlyonitssizeandtheappliedg-force.第8頁，課件共25頁，創(chuàng)作于2023年2月SizeofmajorcellorganellesNucleus 4-12mPlasmamembranesheets 3-20mGolgitubules 1-2mMitochondria 0.4-2.5mLysosomes/peroxisomes 0.4-0.8mMicrosomalvesicles 0.05-0.3m第9頁，課件共25頁，創(chuàng)作于2023年2月Differentialcentrifugationofatissuehomogenate(I)1000g/10minDecantsupernatant3000g/10minetc.第10頁，課件共25頁，創(chuàng)作于2023年2月Differentialcentrifugationofatissuehomogenate(II)Homogenate–1000gfor10minSupernatantfrom1–3000gfor10minSupernatantfrom2–15,000gfor15minSupernatantfrom3–100,000gfor45minPellet1–nuclearPellet2–“heavy”mitochondrialPellet3–“l(fā)ight”mitochondrialPellet4–microsomal第11頁，課件共25頁，創(chuàng)作于2023年2月Differentialcentrifugation(III)

Expectedcontentofpellets1000gpellet:nuclei,plasmamembranesheets3000gpellet:largemitochondria,Golgitubules15,000gpellet:smallmitochondria,lysosomes,peroxisomes100,000gpellet:microsomes第12頁，課件共25頁，創(chuàng)作于2023年2月Differentialcentrifugation(IV)Poorresolutionandrecoverybecauseof:ParticlesizeheterogeneityParticlesstartingoutatrminhavefurthesttotravelbutinitiallyexperiencelowestRCFSmallerparticlesclosetormaxhaveonlyashortdistancetotravelandexperiencethehighestRCF第13頁，課件共25頁，創(chuàng)作于2023年2月Differentialcentrifugation(V)Fixed-anglerotor:Shortersedimentationpathlengthgmax>gminSwinging-bucketrotor:Longsedimentationpathlengthgmax>>>gmin第14頁，課件共25頁，創(chuàng)作于2023年2月Differentialcentrifugation(VI)RateofsedimentationcanbemodulatedbyparticledensityNucleihaveanunusuallyrapidsedimentationratebecauseoftheirsizeANDhighdensityGolgitubulesdonotsedimentat3000g,inspiteoftheirsize:theyhaveanunusuallylowsedimentationratebecauseoftheirverylowdensity:(

p-l)becomesratelimiting.第15頁，課件共25頁，創(chuàng)作于2023年2月DensityBarrierDiscontinuousContinuousDensitygradientcentrifugation第16頁，課件共25頁，創(chuàng)作于2023年2月Howdoesagradientseparatedifferentparticles?LeastdenseMostdense第17頁，課件共25頁，創(chuàng)作于2023年2月Whenp>l:vis+veWhenp=l:vis0Predictionsfromequation(I)第18頁，課件共25頁，創(chuàng)作于2023年2月Whenp<l:vis-vePredictionsfromequation(II)第19頁，課件共25頁，創(chuàng)作于2023年2月SummaryofpreviousslidesAparticlewillsedimentthroughasolutionifparticledensity>solutiondensityIfparticledensity<solutiondensity,particlewillfloatthroughsolutionWhenparticledensity=solutiondensitytheparticlestopsedimentingorfloating第20頁，課件共25頁，創(chuàng)作于2023年2月Buoyantdensitybanding

Equilibriumdensitybanding

Isopycnicbanding15234第21頁，課件共25頁，創(chuàng)作于2023年2月1233Formatsforseparationofparticlesaccording

totheirdensityWhendensityofparticle<densityofliquidVis-ve第22頁，課件共25頁，創(chuàng)作于2023年2月DiscontinuousResolutionofdensitygradientsContin