Biology Reference
In-Depth Information
different tissues results in preparations typically with 92-98 %
purity on a protein basis [
6
]. The intensity and duration of homog-
enization are the critical factors in determining the trade-off
between intactness versus quantity of the isolated mitochondria.
The choice of homogenization technique depends upon the rigid-
ity of the tissue under study. Generally, the more rigid the tissue,
the more shearing forces required for tissue disruption and this is
best provided by mechanical devices such as a Polytron
®
homoge-
nizer. This suits fi brous tissues such as rice or wheat shoots whereas
pliable tissues such as Arabidopsis rosettes and spinach leaves can
be homogenized by hand using a mortar and pestle. The optimal
ratio of homogenization medium to plant tissue is generally 1 g of
tissue to 4-6 mL of homogenizing solution. Altering this ratio may
reduce quality or yield. All buffers and equipment should be ice-
cold before beginning the isolation.
3.2.1 Homogenization
1. If using mortar and pestle, grind the tissue in homogenization
medium using a mixture of downward and circular strokes.
The addition of acid-washed sand may enhance the rupturing
of tissue.
2. If using Polytron
®
homogenizer, blend the tissue in a Perspex
chamber with homogenization medium at 50 % full speed
with a succession of 1-s bursts. We generally fi nd that three to
fi ve bursts give the appropriate amount of homogenization.
Too many bursts will increase yield, but may also decrease the
quality of the isolated mitochondria.
Before mitochondria can be enriched by differential centrifugation,
particulate matter and cell debris need to be removed by fi ltration.
A wide range of fabrics are employed for fi ltration; we routinely use
between two and four layers of Miracloth (
see
Note 16
). For cen-
trifugation any centrifuge capable of the desired relative centrifugal
force with a fi xed-angle rotor with suffi cient capacity (~400 mL) for
the homogenate will suffi ce. We generally use a Beckman Coulter
J-26XP centrifuge with a Beckman Coulter JA-25.50 rotor (
see
Note 17
). It is important to conduct these initial fi ltration and
centrifugation steps as quickly as possible, to reduce the time that
mitochondria are exposed to the damaging vacuole compounds
released during homogenization and present in the initial lysate.
1. The homogenate is fi ltered through four layers of Miracloth
via a funnel into conical fl ask. The speed and yield of this pro-
cess are increased by gently wringing the cloth into the funnel.
This is best performed in a 4 °C cold room.
2. The fi ltrated homogenate is transferred into 50 mL centrifuge
tubes, and centrifuged in a precooled rotor for 5 min at
1,500 ×
g
at 4 °C.
3.2.2 Filtering and
Differential Centrifugation
