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3cell fractionation

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3cell fractionation

  1. 1. cell fractionation-----centrifugation
  2. 2. Contents1.Principle i. Basic principle ii. Centrifugation2.procedure
  3. 3. 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.
  4. 4. Centrifugation•Goal: to separate kinds of cellularorganelles and large molecules•Equipment: centrifuge•Classification: i : Differential centrifugation ii: Density gradient centrifugation•Analysis: cellular and biochemistry tomeasure in quality and quantity
  5. 5. 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
  6. 6. Sedimentation sequenceCell homogenate Whole cell Mitochondria microsomes Ribosomes, nucleus lysosomes small vesicles Virus, peroxisomes Large macro- cytosome molecules Speed low high : Size: large small
  7. 7. The preparative ultracentrifuge
  8. 8. Part 2 . procedure 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 times , Put into 3 ml sucrose(0.25M) ,Cut into small pieces , Homogenate for 5 to 8 times , Filter into centrifuge tube of 10 ml through six-layer gauze Smear 1 Put 1 ml to ependorf tube(A) , centrifugate for 10min at 3000rpm precipitation supernatant liquid
  9. 9. precipitation supernatant liquid smear2 Put supernatantMake precipitation the into liquid to dorfsuspension with 1ml sucrose tube(B)(1M) 12000rpm,20min 3800rpm,10min Dispose supernatant liquid , make the precipitation into Dispose supernatant suspension with 1ml liquid , make the sucrose(0.25M) precipitation into suspension with a little sucrose(0.25M) 12000rpm,20min precipitation supernatant liquid smear3 make the precipitation into smear4 smear5 suspension with a little sucrose(0.25M)
  10. 10. 1.Dye the nucleic acid 2.Dye the mitochondria Smear 1 , 2 , 3 Smear 4 , 5Fix in 95% alcohol for Dye in Janus green B5min , open-air dry for 15 minDye in methyl green–pyronin for 20min , wash wash , dry , observ e Wet in the acetone for 2 or 3 times wash , dry , observ e
  11. 11. 将推片自血滴左 侧向右移动当血滴均匀地附着在两片之间后,再将推 片向左平稳地推移 将推片自血滴左 侧向右移动
  12. 12. homework :1. What are the differences of 3 smears of nuclei ?and reasons?2. What are the differences of 2 smears of mitochondria ?and reasons?
  13. 13. After experimentsExperimental 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.
  14. 14. Thank you!

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