Biology Reference
In-Depth Information
2
Materials
The procedure for the isolation of plant mitochondria contains
steps of homogenization, differential centrifugation, and isopycnic
centrifugation, while in some cases FFE is applied as a second
dimension of purifi cation based on surface charge (Fig.
1
). In this
chapter, the FFE procedure is not described as the hardware
required is not widely available and we have chosen to focus on
techniques accessible to most laboratories (for further information
on FFE isolation of mitochondria,
see
refs.
9
,
17
,
28
).
2.1 Preparation of
Density Gradients for
Mitochondrial
Isolation
1. Gradient buffer 1: 20 mM TES-KOH, pH 7.5, 0.6 M man-
nitol, 0.2 % (w/v) BSA.
2. Gradient buffer 2: 20 mM TES-KOH, pH 7.5, 0.6 M sucrose,
0.2 % (w/v) BSA.
3. Percoll
®
(GE Healthcare Life Sciences).
4. Ice bucket with 2 L of ice.
5. 25 mL syringe with a 19-gauge hypodermic needle.
6. Rack for the syringe.
7. Two-chamber gradient pourer and peristaltic gradient pump
(for isolation of mitochondria from green tissues).
2.2 Mitochondrial
Isolation
1. Plant tissue—Mitochondria have been isolated to varying
degrees of purity from a wide variety of plants and plant tis-
sues. When choosing plant material for mitochondrial isola-
tions it is important to choose a tissue that can be produced in
large quantities (>50 g), is amenable to homogenization by
either blending techniques (i.e., Polytron or Waring Blender)
or grinding in a mortar and pestle, and contains low levels of
interfering compounds such as phenolics; particular tissue
types are advantageous in this regard:
Nonphotosynthetic tissues, such as roots, hypocotyls,
tubers, and cell cultures, can be used for large-scale, but
low-percentage-yield, mitochondrial isolation (approxi-
mately 300 mg mitochondrial protein from 5 to 10 kg
fresh weight (FW), 30-60
●
μ
g/g FW) [
1
,
2
,
6
,
7
,
21
,
24
].
Etiolated seedling tissues such as dark-grown cotyledons
or shoots produce high yields of functional mitochondria
because they are free of dense chloroplast membranes
with lower phenolic content than green tissues (approxi-
mately 20 mg mitochondrial protein from 100 g FW,
200
●
μ
g/g FW) [
28
,
45
].
Green tissues, such as leaves, are used for mitochondria iso-
lation for photosynthetic-related function analysis, although
the high abundance of thylakoid membranes and phenolics
●
