Contents1.Principle i. Basic principle ii. Centrifugation2.procedure
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.
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
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 homogenate Whole cell Mitochondria microsomes Ribosomes, nucleus lysosomes small vesicles Virus, peroxisomes Large macro- cytosome molecules Speed low high : Size: large small
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
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)
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