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BIOC23: Lec 2 - Cell fractionation and centrifugation.doc

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Biological Sciences
Rongmin Zhao

Lec 2 Cell fractionation and centrifugation Cellular components and macromocs are temporally and spatiallyrearrangedCell fractionation separate heterogenous cellular components into homogenous setsIf youve purified a whole bunch of proteins but you are actually looking for ONE specific proteinuse technique like SDSPAGE to get high purity of this specific proteinoAfter purifying proteinscan conduct structural and fxn l studyoCan use these protein in other experimentsCell fractionation stepso1 homogenizationbreaking cellsOsmotic alterationPhysical forceCells w cell wallsFreezethaw methodprotective reagent 10 glycerolDMSOprotects membrane so that cell will remain intactFrench pressureapplied high pressure to cello2 filtration optremove unbroken cellsunwanted tissueso3 purificationseparate diff components by differential centrifugationo4 further protein purification opt based on specific protein propertiesIon exchangeGel filtrationAffinityOnly do differential centrifugation if you want to purify some protein thats trapped WITHIN organelle or membrane vesiclesWhy centrifugation can separate diff particles3 forces applied to particles during centrifugationoFb buoyancyoFd viscous dragfrictionoFc centrifugal forceWhen you get near the bottom the particle sinks at constant speed bc all the forces cancel each other outUnder centrifugation force the forces working on any particle will rapidly reach equilibriumFcFdFb0 So for a given particle it will move to the bottom at a constant speed Particles w diff sedimentation coefficient will move w diff speeds oThe sedimentation coefficient s and therefore the speed is dependent onMmolar massffriction ratioparticle shape psolution density
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