Cell Fractionation by Differential Centrifugation
2007-08 INS Winter Quarter – Lab 2
The purification of a specific macromolecule often begins with the isolation of a particular cellular organelle. For
example, if we were interested in studying the organization of chromatin in yeast cells, we could start by
isolating nuclei.
In a cell fractionation procedure, the cell must first be disrupted by breaching the cell boundaries. Common
methods include grinding in a mortar, homogenization in ground glass, and exposure to sonic vibrations. In
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most cases, disruption is carried out at low temperatures (between 0 and 4 C) to minimize proteolysis and loss
of biological activity. After cell disruption, the particulate components are separated from each other by high
speed centrifugation.
In differential centrifugation, the heavier particles settle first, followed by gradual separation of lighter particles by
sedimentation. For example, centrifugation at a speed 600 times gravity of a tissue homogenate suspended in
0.25 M sucrose results in the separation of nuclei. If the supernatant resulting from this centrifugation is
centrifuged at 10,000 times gravity, the mitochondria are pelleted out of solution. Centrifugation at still higher
speeds (100,000 times gravity) pellets other particulate components, such as the microsomal fraction
(endoplasmic reticulum).
[The relative centrifugal force (RCF) is expressed by using g units, where 1 g is the force of the earth’s gravity.
The force exerted on a particle in a centrifuge is expressed by the following equation:
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2
RCF = 1.119 x 10 (rpm ) r
where rpm is the revolutions per minute of the rotor and r is the distance (in cm) of the particle from the axis of
rotation.]
Resolution of cellular structures under the light microscope can be increased by fixation and staining of tissue
samples. Fixation involves the selective preservation of morphological organization and chemical content for
microscopic observation. The most reliable fixatives contain one or more substances that precipitate the
proteins of the cell or render them insoluble without precipitating them. Staining involves chemical reactions
between a dye and proteins or nucleic acids. The selection of a specific stain depends on a number of factors
including the nature of the material to be studied, the type and pH of the fixative used, and the chemical
reactivity of the dye.
We will be using two stains in our experiments today: aceto-orcein and Janus green. Aceto-orcein specifically
stains nuclei. It contains a fixative, so both staining and fixing are achieved in one step. Janus Green B
specifically stains mitochondria, and is an example of a substance known as a vital stain. When Janus Green
is introduced into a cell, it stains all of the cell components green, since it is maintained in its oxidized state.
After a brief time, however, it is reduced to its colorless form in all parts of the cell except the mitochondria,
where it is reoxidized by cytochrome oxidase (an enzyme that takes part in the final stages of cellular
respiration) and retains its color.
Laboratory aims
In the laboratory, you should generate fractions enriched for nuclei and mitochondria, starting from a cauliflower
cell homogenate.
After completing this lab, you should understand 1) the principles and methods of tissue homogenization and
differentialcentrifugation; and 2) how organelles can be visualized through the use of specific chemical stains.
Protocol
Overall strategy
This lab is aimed at isolating a nuclear fraction and mitochondrial fraction from cauliflower cells using the
method of differential centrifugation. Cauliflower tissue will be homogenized in a mortar and pestle with a
buffered, isotonic sucrose solution and a quantity of sand. The homogenate will first be filtered through
cheesecloth to remove larger debris and then centrifuged at a relatively low speed to pellet whole cells and the
nuclear fraction. The supernatant will then be centrifuged at a relatively high speed to pellet the mitochondrial
fraction. The starting material and each fraction will be reacted with chemical stains that will allow better
visualization of these organelles under the compound microscope.
Reagents
Homogenization buffer: 0.4 M sucrose, 50 mM potassium phosphate pH 7.7, 5 mM EGTA
Resuspension buffer: 20 mM potassium phosphate, pH 7.0, 20 mM 2-mercaptoethanol
Part A: Homogenization and cell fractionation
Important: Work on ice for all steps!
1. Chill cauliflower heads (the heads should be firm and tight, not in the flowering or “loose” stage). Carefully
use a razor blade to cut the outer 5-10 mm onto aluminum foil over ice. Begin with about 5 g of cauliflower.
2. Using a chilled mortar and pestle, grind the cauliflower with 2.5 g clean sand for 30 sec. Add 15 ml of
homogenization buffer and grind an additional 30 sec to suspend the mixture. Pour off into a 50 ml plastic
centrifuge tube. Rinse the mortar with 5 ml of buffer and combine it in the centrifuge tube with the original
mixture. Let the sand settle to the bottom for 2 min on ice.
3. Filter this mixture through four layers (2 pieces) of cheesecloth into a chilled, 30 ml centrifuge tube. Using
gloves, squeeze fluid from cheesecloth into the tube. This is your INITIAL HOMOGENATE. Save a 0.5 ml aliquot of
the homogenate in a labelled Eppendorf tube.
4. Using a balance, match your tube with another group’s tube to within 0.1 g. Centrifuge at 600 xg for 10 min
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at 4 C.
5. Decant the supernatant into a clean, chilled 30 ml centrifuge tube. The pellet is your NUCLEAR PELLET. Keep
the nuclear pellet on ice.
6. Match your group’s tube with another group’s tube to within 0.1 g. Centrifuge the post-nuclear supernatant at
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10,000 xg for 15 min at 4 C.
7. Save 0.5 ml of the FINAL SUPERNATANT into a labelled Eppendorf tube (it should be fairly clear). Discard the
remainder of the supernatant. The pellet is your MITOCHONDRIAL PELLET. Keep the mitochondrial pellet on ice.
8.
a)
b)
c)
d)
At the end of this procedure, you should have four fractions:
0.5 ml of the INITIAL HOMOGENATE
NUCLEAR PELLET
MITOCHONDRIAL PELLET
0.5 ml of the FINAL SUPERNATANT
Add 2.0 ml of the ice-cold resuspension buffer to the MITOCHONDRIAL PELLET. Scrape the pellet from the wall of
the tube with a clean spatula and resuspend by vortexing.
Part B: Characterization by microscopy
Examination without staining
First examine each of your four fractions under 400X magnification without staining.
Staining and analysis of the nuclear fraction
Remove a small bit of the nuclear pellet with a spatula and smear on a clean slide. Immediately add several
drops of aceto-orcein. After 15 sec, add a coverslip and gently press out the excess stain with a Kimwipe.
Examine the preparation under 400X magnification.
Identify nuclei in this fraction and measure the size of 5 nuclei. You may be able to observe chromosomes by
examining the slide under 1000X magnification.
Now look for the presence of nuclei in each of the three other fractions. What can you conclude about relative
enrichment?
Staining and analysis of the mitochondrial fraction
Place 200 µl of the mitochondrial suspension in an Eppendorf tube. Add an equal volume of the Janus green
dye and vortex gently. Let the mixture incubate at room temperature for 10 min.
Put one drop of this mixture onto a clean slide. Add a coverslip and examine under 400X magnification.
Examine the same slide under 1000X magnification using oil immersion.
Identify mitochondria in this fraction and measure the size of 5 mitochondria.
Now look for the presence of mitochondria in each of the three other fractions. What can you conclude about
relative enrichment?
Pre-lab questions
After the first spin, what is in the nuclear pellet? What is in the post-nuclear supernatant?
After the second spin, what is in the mitochondrial pellet? What is in the post-mitochondrial supernatant?
Post-lab question
Explain the role of each component in the homogenization buffer and in the resuspension buffer.
Background reading on centrifugation
Freeman, p. 140.
References
Cohn, N.S. (1969). Elements of Cytology (Second Edition). Harcourt, Brace and World, Inc.
Randall, D.D. (1982). Pyruvate dehydrogenase complex from broccoli and cauliflower. In Methods in
Enzymology 89, 408-414.