Biomedical Engineering Reference
In-Depth Information
9. [
35
S]-labeled mitochondrial precursor protein prepared in the
in vitro translation reaction. Keep in single-use aliquots of
12 ʼL at −80 °C (
see
Note 14
).
10. Isolated yeast mitochondria at 10 mg/mL kept in single-use
aliquots at −80 °C (
see
Note 15
).
3
Methods
3.1
Yeast Culture
Yeast cells are grown in Erlenmeyer flasks in liquid cultures under
constant agitation (approx. 140 rpm). The optimal growth tem-
perature for
S. cerevisiae
is 30 °C; however, temperature sensitive
mutants should be grown at 25 or 15 °C. The growth rate depends
notably on the yeast strain. Therefore, it is recommended to mea-
sure the generation time for each strain before inoculating the
scaled up culture that will be used for isolation of mitochondria (
see
Note 2
).
1. Inoculate 50 mL of culture with the strain of interest.
2. Follow the growth of the strain over several generations
under the same conditions. Take care that it should always
be kept at the logarithmic growth phase (OD
600
should
never exceed 2.0).
3. Measure regularly the OD
600
of the culture, and calculate the
generation time of the strain by using the following formula:
=
(
)
(
)
generation time
t
*log /log
2
OD final OD initial=
/
600
600
where
t
is the time of growth in hours.
4. Inoculate the final culture for the isolation of mitochondria
considering the generation time and expecting an OD
600
of 1.5
at the time of harvesting. The cultures must be in the logarith-
mic growth phase at the moment of harvest. The amount of
culture inoculated depends exclusively on the experimental
needs, usually from 10 L of a culture with an OD
600
of 1.5
approx. 20 g of cells (wet weight) are obtained. The mitochon-
drial yield from this amount of cells is approx. 20 mg.
1. Grow the desired amount of yeasts to an OD
600
of 0.8-2.0.
2. Collect the cells at 3,000 ×
g
for 5 min and determine the wet
weight.
3. Resuspend the pellet in 100 mL distilled water and centrifuge
at 3,000 ×
g
for 5 min.
4. Resuspend the cells in MP1 buffer (2 mL/g of wet weight)
and incubate the suspension for 10 min at 30 °C under agita-
tion (approx. 140 rpm).
3.2 Isolation
of Mitochondria by
Differential
Centrifugation
