3. Basic 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.
4. Centrifugation
•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
5. Differential 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
6. Sedimentation sequence
Cell homogenate Whole cell Mitochondria microsomes Ribosomes,
nucleus lysosomes small vesicles Virus,
peroxisomes Large macro-
cytosome
molecules
Speed low high
:
Size: large small
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. precipitation supernatant liquid
smear2
Put supernatant
Make precipitation the into
liquid to dorf
suspension 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. 1.Dye the nucleic acid 2.Dye the mitochondria
Smear 1 ， 2 ， 3 Smear 4 ，
5
Fix in 95% alcohol for
Dye in Janus green B
5min ， open-air dry
for 15 min
Dye in methyl green–
pyronin for 20min ， wash wash ， dry ， observ
e
Wet in the acetone
for 2 or 3 times
wash ， dry ， observ
e
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. 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.
