BIO 330 Cell Biology
Spring 2017
Laboratory
Chloroplast and Mitochondrial Isolation and Assay
Background
The study of cell biology involves understanding the role of particular organelles within the cell. To study
particular organelles, it is helpful to be able to produce tissue homogenates which are enriched in the
organelle you want to study. Differential centrifugation is a technique that takes advantage of the
varying densities of organelles in order to separate one type of organelle from another. Larger
structures tend to move to the bottom of a tube more quickly than smaller structures. Therefore,
shorter, slower centrifugation can pellet large structures like nuclei and whole cells; while longer, faster
centrifugation pellets smaller structures like mitochondria or even lysosomes.
During centrifugation, it is not RPM (rotations per minute) that matters; rather, it is RCF (rotational
centrifugal force) * minute. RCF = [(rpm*2)/ 60]2*r/g, where r = radius of rotor in cm, and g is the
gravitational constant, 980cm/s2. For example, 4200 RPM in an SS34 rotor yields 1 X 103 RCF. In our
microcentrifuge, the 3500 RPM yields the same RCF. Furthermore, because it is RCF*min that matters,
as opposed to simply RCF, a centrifuge that cannot spin as fast as the protocol requires can still be used,
if a sample is spun longer.
For organelles that are very similar in density or mass, differential centrifugation on its own may not be
sufficient to separate them. In these cases, layering the sample above a solution that creates a density
gradient within the tube can be useful. For example, a researcher can create several different sucrose
concentrations; they would put the densest layer at the bottom, and the least dense at the top. The
organelles will travel down the tube until their density matches the density of a particular layer. The
researcher can then simply pipette the matching layer into a new tube to have an enriched preparation
of the target organelle.
Chloroplasts and mitochondria are important for cells’ energy needs. Mitochondria are found in every
eukaryotic cell; they metabolize organic molecules to produce ATP, producing carbon dioxide as a
byproduct. Chloroplasts are found in the green parts of a plant, and use light and carbon dioxide to
make organic molecules; these organelles produce oxygen as a byproduct of photosynthesis. Although
these two organelles function in very similar ways, chloroplasts are much larger than mitochondria.
Thus, they are relatively easy to separate by differential centrifugation. Today, after isolating
mitochondria and chloroplasts, we will assay their presence by measuring evolution of carbon dioxide or
oxygen, respectively.
Methods
Take note that today’s protocol will involve long periods of waiting for particular steps. In order to finish
lab in a reasonable amount of time, you will need to move on to later steps of the protocol while earlier
steps are in process. Plan accordingly.
1. Prepare a spinach homogenate, with the whole class working together:
a. Pack spinach leaves into a blender to a depth of about 1-2 inches.
b. Add 150 mL of 0.5M sucrose solution (an osmotically balanced buffer).
BIO 330 Cell Biology
Spring 2017
Laboratory
c. Homogenize spinach by running blender at top speed for 5 min in 15-second bursts separated
by 15 seconds of rest.
d. Pour through 4 layers of cheesecloth into an ice-cold flask using a pre-chilled funnel.
e. Transfer liquid to 4 50-mL centrifuge tubes; balance tubes to within 0.1g and place in the
prechilled tabletop centrifuge.
f. Centrifuge at 500g for 2 min to clear out large tissue pieces, and transfer 14 mL of
homogenate to 4 new 15-mL conical centrifuge tubes pouring carefully (do not disturb the
pellet). Each group will now “own” one of the 4 15-mL tubes.
g. Make a wet mount using ~50 L of well-mixed homogenate. Make a drawing in your
notebook that will allow you to track the relative number of chloroplasts in each step so that
you can tell you are producing more enriched samples.
2. Differential centrifugation of homogenate (each group working separately here and following,
although centrifugation steps will need to be done at the same time to ensure all groups get finished
within lab time):
a. Centrifuge homogenate at 1500xg for 10 min to pellet chloroplasts. Use a 5-mL pipette to
carefully transfer all of the supernatant to a new 15-mL conical centrifuge tube. The supernatant
will be used for isolation of mitochondria. Label your tubes accordingly. Keep chloroplast on ice
while starting mitochondrial centrifugation.
b. Centrifuge mitochondrial supernatant tube at 3000xg for 15 min.
c. During mitochondrial centrifugation, resuspend chloroplast pellet in 1.4 mL of phosphate
buffer by very gently pipetting up and down with a 1000-L pipette. Make a wet mount as
before; draw a record in your notebook. (Hopefully the density of chloroplasts will be much
higher now.) Keep tube on ice until assay.
d. When mitochondrial supernatant tube is finished, transfer 1.0 mL of supernatant to each of 6
microcentrifuge tubes and centrifuge at 18,000 x g for 20 minutes. The pellets now contain
mitochondria. Transfer pellets, combining into a single new 15-mL round-bottom tube (label it),
and resuspend in 0.6 mL of phosphate buffer. (Note: You can use the phosphate buffer to collect
the pellets if necessary.) Make a wet mount; record appearance (may not be able to see
mitochondria at all due to size; you are really looking for absence of chloroplasts). Discard
supernatant.
3. Oxygen evolution assay: Because chloroplasts produce O2 as a byproduct of photosynthesis, we can
use O2 concentration as an index of chloroplast function. We will compare chloroplasts in the presence
of light with those in the absence of light to show that changes in O2 over time really are due to
photosynthesis.
a. Launch LoggerPro software. Calibrate O2 sensor as shown by your instructor.
b. Cover a plastic flask with aluminum foil. Pipette 1 mL of chloroplast solution into the flask,
and insert an O2 probe tightly.
c. Click the green Collect button. Let the run go for the entire 15 minutes. The run will end on its
own. When prompted, click Save Latest Run and continue.
d. Uncover the flask, shine a bright light on the flask, then collect another 15 minute run.
BIO 330 Cell Biology
Spring 2017
Laboratory
e. Analyze your data: record the overall slope of each run. If there are “weird” or unstable areas
near the beginning of each run, you can exclude these (ask instructor for help). Be sure to record
appropriate units for the slope.
f. Repeat this assay using the mitochondrial preparation if time allows.
4. Carbon dioxide evolution assay: Because mitochondria produce CO2 as a byproduct of aerobic
respiration, we can use CO2 concentration as an index of mitochondrial function. We will compare
mitochondria with and without added pyruvate (or another carbon source metabolizable by the Krebs
cycle).
a. Launch LoggerPro software. Calibrate CO2 sensor as shown by your instructor.
b. Pipette 1 mL of mitochondria solution into a plastic flask, then insert a CO2 probe tightly.
c. Click the green Collect button. Let the run go for the entire 15 minutes. The run will end on its
own. When prompted, click Save Latest Run and continue.
d. Add 10 L of pyruvate to the solution in the flask, then collect another 15 minute run.
e. Analyze your data: record the overall slope of each run. If there are “weird” or unstable areas
near the beginning of each run, you can exclude these (ask instructor for help). Be sure to record
appropriate units for the slope.
f. Repeat this assay using the chloroplast preparation if time allows.
Results
You should have the O2 and/or CO2 production rates for each preparation and in each condition
recorded and clearly labeled in your notebook.
Drawings of chloroplast enrichment steps should be labeled in your notebook.
Conclusions / Discussion
Describe what your results say about your centrifugation preparations. How pure are they?
What is the purpose of using a sucrose solution to homogenize tissue?
Describe how centrifugation is able to separate subcellular structures. What is generally the lower limit
of what can be separated by centrifugation? What other centrifugation-based techniques can be used
to study particular cellular components? You will need to do some extra research to answer these
questions.
Note: The protocol is derived from an article published by Lang et al. in Plant Cell Reports, 2011; 30(2):
205-215.