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!