3cell fractionation

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  • cell fractionation

    -----centrifugation

  • ContentsPrinciple i. Basic principle ii. Centrifugationprocedure

  • Basic principleFact1:The structures in the cell with different specific gravities and sizes have various sedimentation velocities in the same centrifugate field.Fact2:We can use different mediums or velocities in centrifugation to separate different structures one by one according to the fact 1.

  • CentrifugationGoal: to separate kinds of cellular organelles and large moleculesEquipment: centrifugeClassification: i : Differential centrifugation ii: Density gradient centrifugationAnalysis: cellular and biochemistry to measure in quality and quantity

  • Differential centrifugationFeature:1.uniform density of medium 2.progressively higher velocityUsage: separate subcellular structure or orangells with great disparity in sizeSedimentation sequence: large to smallMethod: homogenate ,speed up progressively and centrifugate one by one

  • Sedimentation sequenceCell homogenateWhole cellnucleus cytosomeMitochondrialysosomes peroxisomesmicrosomessmall vesiclesRibosomes,Virus,Large macro-moleculesSpeed :lowhighSize:largesmall

  • The preparative ultracentrifuge

  • Execute the mouse with dislocation ,Take out the liver from abdominal cavity , Cut into trunks , Wash Take half of the liver ,Wash in sucrose(0.25M) for three timesPut into 3 ml sucrose(0.25M) ,Cut into small piecesHomogenate for 5 to 8 timesFilter into centrifuge tube of 10 ml through six-layer gauzePut 1 ml to ependorf tube(A) centrifugate for 10min at 3000rpmSmear 1precipitationsupernatant liquidPart 2 . procedure

  • Make precipitation the into suspension with 1ml sucrose (1M)3800rpm,10minDispose supernatant liquid make the precipitation into suspension with a little sucrose(0.25M)precipitationsmear3supernatant liquidPut supernatant liquid to dorf tube(B)smear212000rpm,20minDispose supernatant liquid make the precipitation into suspension with 1ml sucrose(0.25M)12000rpm,20minprecipitationsupernatant liquidmake the precipitation into suspension with a little sucrose(0.25M)smear5smear4

  • 1.Dye the nucleic acid2.Dye the mitochondria

  • homework1. What are the differences of 3 smears of nuclei ?and reasons?2. What are the differences of 2 smears of mitochondria ?and reasons?

  • After experimentsExperimental appliance should be rinsed;Slides and burettes should be taken to the basin;Animals (after experiment) should be taken to the bag;Sweep the desks and floor.

  • Thank you!