Isolation of Mitochondria and Analysis of Subcellular Fractions

SLE206 CELL BIOLOGY 1
Isolation of Mitochondria and Analysis of Subcellular Fractions.
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SLE206 CELL BIOLOGY 2
Introduction
Mitochondria are eukaryotic cell organelles that are known for harboring pathways in
ATP synthesis through phosphorylation and tricarboxylic acid cycles(Schrepfer and
Scorrano, 2016). Mitochondria has two membranes which contain unusual lipid cardiolipin
and very rich in proteins. The outer membrane has porins which allow molecules exchange
between the cytoplasm and intermediate space(Rizzuto et al., 2012). However, the inner
membrane is completely impermeable even to small molecules except for water carbon IV
oxide and oxygen. In addition, the inner membranes allow some other molecules such as
pyruvate and succinate(Ortiz-Espín et al., 2017). There are various transporters in the inner
membranes which ensure import and exports of crucial metaborates. The major function of
the inner membranes is to aid the transport of respiratory chain complexes, ATP synthase,
and other enzymes. The inner part of the inner membrane on the side of the matrix contains
enzymes for oxidative phosphorylation and electron transport chains(Nunnari and
Suomalainen, 2012). The matrix is rich in proteins and contains all the citric acid cycle
enzymes except the succinate dehydrogenase.
Isolation of mitochondria is crucial in the modern field of medicine and research in
essence that it helps in the understanding of structure and function of the cells as well as
cancer and metabolic diseases(Schmitt and Zischka, 2018). For instance, cancer cells exhibit
several mitochondrial dysfunctions including increase transmembrane potential, change in
energy metabolism and elevated ROS generation. Therefore, these changes provide scientists
with referential target cancer cells mitochondria hence improving the selectivity of various
therapies(Dimauro et al., 2012). Separation of cellular components is usually done using
subcellular fractionation which normally uses one or properties of each organelle or cellular
compartment including buoyancy density, shape and size, and surface change density basing
on differential centrifugation in media of high viscosity(Schrepfer and Scorrano, 2016). The

SLE206 CELL BIOLOGY 3
isolation of mitochondria from subcellular structures using differential centrifugation
methods normally relies on various elements(Dimauro et al., 2012). These include
mechanical rapturing of cells, low-speed differential centrifugation to remove large
organelles structures and debris and finally isolating mitochondria using higher speeds to
isolate mitochondria(Schmitt and Zischka, 2018). To determine which fraction has the
highest concentration of mitochondria or whether the process of the isolation was successful,
a specific marker for mitochondria succinate dehydrogenase activity is tested(Azimzadeh et
al., 2016).
Aims of the study.
This practical study seeks to isolate mitochondria fractions from the liver and carry out
differential centrifugation on the liver extracts. Besides, the succinate hydrogen activity of all
extract isolated from the liver will be evaluated to determine the concentration of
mitochondria in various fractions.
Methods.
The experiment procedure will consist of three major steps which include subcellular
fractionation to isolate mitochondria from liver cell lyse, BCA assay and proteins calculation
and finally the confirmation of mitochondria fractionation using the SDH essay. The methods
and procedures of the experiments are explained below.
Step1; Subcellular fractionation of mitochondria from the liver.
Prelab preparation sample prepared by a technical staff.
During the pre-preparation phase, technical staff will obtain samples of fresh liver pieces
weighing around 500 mg and ad equivalent volumes of cry-storage solution. The cell tissues
will then be snap-frozen and store in liquid nitrogen for about three months. During the day

SLE206 CELL BIOLOGY 4
of the experiment, the thawing medium will be warmed using 45 decrees water bath for
thawing frozen tissues. After that, the frozen tissues will be thawed by adding 10um for every
2.5mg of tissue. The technical staff will then dice 2mm pieces into a 50ml conical centrifuge
tube containing an isolation buffer and proteins inhibitors. Samples will be homogenized at
maximum speed and be strafer to 2ml crew cap microfuge tubes for the procedure.
Actual Experiment Procedure
During the experiment, five sterile 2ml screwcaps will be prepared by labeling them
WCL, SN1, SN2, SN3, and Mito on the lids. The screwcaps will also have student initials at
the sides and practical sessions to allow more accurate labeling and stored into the ice. Put
the cell lysate measuring 100ul into tube labeled WCL and store into ice. Pellet the cell debris
in the homogenate at 1000x for around ten minutes at 4 degrees Celcius. After that, the
supernate obtained should be transferred to the tube labeled Mito with a 1 mil filter tip and
the tube with cell pallets be discarded. The supernate in tube labeled Mito should also be
centrifuged for 10 min at 1000xg at a similar temperature and be transferred to tube labeled
SN1. The supernate in SN1 will then be resuspended as demonstrated by the instructor to
prevent cell clamps remains at the base. An additional 1000ul of the isolation buffer should
then be added, mix well and centrifuged at 12,000xg in the same duration and time as above.
Pipette the supernatant in SN! To tube labeled SN2, resuspend and add another 1000ul of
isolation buffer. Repeat the procedure as in SN1 and pipette to the tube labeled SN3. After
that resuspend the pellets in SN3 with 400ul of the isolation buffer, mix and store in the ice.
Store the above sample at -80 decree celsius until the next step of the experiment procedure.
Step 2; BCA Essay and proteins calculations.
During this step, use table 1 below to prepare protein standards in sterile 1.5ml
microcapsule tubes and observe. As demonstrated in table 2, transfer 25ul of each standard in