3cell fractionation
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Transcript of 3cell fractionation
cell fractionationcell fractionation
-----centrifugation-----centrifugation
ContentsContents
1.Principle i. Basic principlei. Basic principle ii. Centrifugation ii. Centrifugation
2.procedure
Basic principleBasic principle
Fact1: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.
CentrifugationCentrifugation•Goal: to separate kinds of cellular organelles and large molecules•Equipment: centrifuge•Classification:
i : Differential centrifugation
ii: Density gradient centrifugation•Analysis: cellular and biochemistry to measure in quality and quantity
Differential centrifugationDifferential centrifugation
Feature:1.uniform density of medium
2.progressively higher velocity
Usage: separate subcellular structure or orangells with great disparity in size
Sedimentation sequence: large to small
Method: homogenate ,speed up progressively and centrifugate one by one
Sedimentation sequenceSedimentation sequence
Cell homogenate Whole cellnucleus cytosome
Mitochondrialysosomes peroxisomes
microsomessmall vesicles
Ribosomes,Virus,Large macro-molecules
Speed :
low high
Size: large small
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 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
Put 1 ml to ependorf tube(A) ,centrifugate for 10min at 3000rpm
Smear 1
precipitation supernatant liquid
Part 2 . procedurePart 2 . procedure
Make precipitation the into suspension with 1ml sucrose (1M)
3800rpm,10min
Dispose supernatant liquid , make the precipitation into suspension with a little sucrose(0.25M)
precipitation
smear3
supernatant liquid
Put supernatant liquid to dorf
tube(B)
smear2
12000rpm,20min
Dispose supernatant liquid ,make the precipitation into suspension with 1ml sucrose(0.25M)
12000rpm,20min
precipitation supernatant liquid
make the precipitation into suspension with a little
sucrose(0.25M)smear5
smear4
1.Dye the1.Dye the nucleic acid 2.Dye the mitochondria2.Dye the mitochondria
Smear 1 , 2 , 3
Fix in 95% alcohol for 5min , open-air dry
Dye in methyl green– pyronin for 20min , wash
Wet in the acetone for 2 or 3 times
wash , dry , observe
Smear 4 ,5
Dye in Janus green B for 15 min
wash , dry , observe
将推片自血滴左 侧向右移动
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
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?
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.
Thank you!