MEMBRANES AND PROTEINS REPORT – THE EXTRACTION AND PURIFICATION OF
MITOCHONDRIA FROM THE MERISTEM OF A CAULIFLOWER
Anne Aloysia Johnson
18975021 THU PM
INTRODUCTION
For details on materials and methods please refer
to the BCH3AAB laboratory manual.
Differential centrifugation relies on the principle of
that different cell types or cellular components
sediment at different velocities. Therefore by
increasing the speed, different components can be
separated from the rest of the mixture (Jeppesen et
al., 2014). This process will be a key component in
the experiment outline in this paper.
RESULTS
The degree of purity of mitochondria can be
gathered from an SDH assay by looking at the
intensity of bands and by whether or not there is
ghosting when the individual bands are considered.
Bright bands and minimal ghosting mean that the
purity is high whereas dull bands and extensive
ghosting can mean that the purity is very low.
Another key component are marker proteins. A
marker protein is a distinguishing protein that will
allow for scientist to differentiate the target
component from all the other components. For
example, this experiment is focused on isolating
the mitochondria and therefore the activity of an
enzyme that is specific to the confinement of the
inner membrane of mitochondria is an excellent
marker. (Alam et al., 2012) So after centrifugation,
both the pellet and the supernatant can be tested for
this specific enzyme activity and the fraction with
higher enzyme activity can be confirmed as the
fraction with the higher concentration of
mitochondria. (Alam et al., 2012)
Finding the amount of protein that was loader into
the mitochondrial extract lane:
Amount of protein in the tube = 0.5mg
0.5mg/100 = 0.005
0.005*0.5mg = 0.0025mg = 25μg
In reference to Figure 1, Lane 6 (total solubilized
mitochondria) and Lane 7 (integral fraction) have
similar bands but Lane 6 has more prominent
bands. Lane 8 (peripheral fraction) has more bands
compared to the other two.
In this particular experiment, due to the interest in
the mitochondria, the marker enzyme that is used
is succinacte dehydrogenase (SDH). SDH is an
enzyme that is found in the inner membrane of the
mitochondria (Jones and Hirst, 2013). In this
experiment, we will be using an SDH assay which
allows for proteins to be denatured before they run
through the gel. This will eliminate any
discrepancies caused by the conformation of a
protein (Jones and Hirst, 2013).
The size of unknown proteins can be estimated by
graphing the relative mobility of markers against
the log of the molecular weight markers (Figure 2).
Then the relative mobility is calculated from which
the molecular weight can be calculated. Thus, the
size of an unknown protein has been estimated.
DISCUSSION
The assay for SDH activity did work according to
the predictions that were made prior to the
commencement of the experiment. Several
controls were used to ensure that the results of the
SDH activity was not affected by other factors.
Some of these controls included malonate (a
competitive inhibitor to SDH), no extract controls
(to ensure when there was no extract present there
was no activity) and no DCPIP controls (to ensure
that none of the other reagents were affecting the
colour and therefore the spectrometric reading).
The major objective of this experiment was to
purify mitochondria from the inflorescence
meristem of the cauliflower. This is was achieved
by using differential centrifugation. In the process
of this experiment, students also learnt about the
relationship between proteins and membranes,
advanced protein analysis techniques and
improved on their quantitive skills.
EXPERIMENTAL PROCEDURES
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The reason for SDH activity in the post
mitochondrial supernatant may be due to the lack
of freshness of the cauliflower and therefore the
mitochondria has decomposed. In this case SDH
will still be present in vesicles. This can confirmed
using a western blot with an SDH antibody.
Major proteins can be located on the gel by
backtracking using the molecular weight of the
band and using the graph (Figure 2). It can be
decided whether a particular protein is integral or
peripheral by the way the bands are spread Ed.
Integral are more detailed.
SDH is a very good marker for intact mitochondria
as it is found in the inner membrane of the
mitochondria and therefore the presence of SDH
will be conjunctive of the presence of
mitochondria.
In conclusion, the aim of this experiment to extract
and purify mitochondria from cauliflower cells
was successful. This process has also allowed for
improving in analytic and quantitative skills in the
lab.
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REFERENCES
Alam, M., Ali, S., Abbasi, A., Kalbacher, H. and Voelter, W. (2012). Design and Synthesis of a PeptidylFRET Substrate for Tumor Marker Enzyme human Matrix Metalloprotease-2 (hMMP-2).
International Journal of Peptide Research and Therapeutics, 18(3), pp.207-215.
Jeppesen, D., Hvam, M., Primdahl-Bengtson, B., Boysen, A., Whitehead, B., Dyrskjøt, L., Ørntoft, T.,
Howard, K. and Ostenfeld, M. (2014). Comparative analysis of discrete exosome fractions
obtained by differential centrifugation. Journal of Extracellular Vesicles, 3(1), p.25011.
Jones, A. and Hirst, J. (2013). A spectrophotometric coupled enzyme assay to measure the activity of
succinate dehydrogenase. Analytical Biochemistry, 442(1), pp.19-23.
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APPENDIX
TUBE
A600nm
15 mins
A600nm
30 mins
∆A600nm
1530mins
∆[DCPIP]
15-30mins
(μM)
(μmoles
DCPIP.min1
)
Volume
of
extracted
assay (ml)
Activity
μmoles.
min-1.ml-1
Specific
activity
μmoles.
min-1.mg-1
Post Nuclear
Supernatant
Post Mitochondrial
Supernatant
Mitochondrial
Fraction
1.04
0.992
0.048
5.34
0.00178
0.5
0.00089
7.05x10-4
0.606
0.606
0
2.88
0.0096
0.5
0.0048
0.00402
1.48
1.37
0.11
8.59
0.00286
0.5
0.00143
0.0028686
Table 1: Summary of data from the mitochondrial purification
1
2
3
4
5
6
7
8
250 kDa
148 kDa
98 kDa
64 kDa
50 kDa
36 kDa
22 kDa
16 kDa
6 kDa
4 kDa
Figure 1: SDS-PAGE gel. This figure shows the SDS-PAGE gel used to determine the purity of
mitochondria in the final samples. In lane 1, 5μl of the molecular weight markers was loaded.
Lane 6 had 5μl of sample total solubilised mitochondria, lane 7 had 5μl of sample integral
membrane protein and lane 8 contains 20μl of sample peripheral membrane protein .
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Graph between Relative Mobility and log of Molecular Weight
of Protein Markers
1,2
Relative Mobility Rf
1
0,8
0,6
0,4
0,2
0
0
0,5
1
1,5
2
2,5
Log of Molecular Weight (kDa)
Figure 2: Relative mobility versus log of the molecular weight of the protein standards.
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