Biomedical Engineering Reference
In-Depth Information
2. Generation of the TOM-TIM-Supercomplex.
Materials are the same as listed in Subheading
2.4
with the
following variations:
(a) Import buffer as described in Subheading
2.4
without BSA.
(b) Methotrexate (MTX): keep as 10 mM solution in 0.1 M
MOPS-KOH, pH 7.2, store at −20 °C, stable for years;
dilute in import buffer w/o BSA as indicated.
2.7 Determination
of Matrix-Directed
Import Forces Exerted
by Hsp70
1. Isolated mitochondria in SEM buffer: 10 mg/ml in protein
content, freshly prepared or thawed
on ice
.
2. Import buffer:
see
Subheading
2.4
.
3. 0.1 mM Methotrexate in import buffer w/o BSA, store at
−20 °C, stable for years.
4. 0.2 M NADH, prepare fresh, keep on ice.
5. 0.2 M ATP (
see
Subheading
2.4
).
6. 1 U/
l apyrase, store at −20 °C, stable for years.
7. 10 mM oligomycin (
see
Subheading
2.4
).
8. ATP-regenerating system (
see
Subheading
2.6
).
9. Buffer R1 (−ATP): 20
ʼ
ʼ
M oligomycin, 5
ʼ
M MTX, in P80
w/o BSA, prepare fresh.
10. Buffer R2 (+ATP): 2 mM ATP, 20 mM creatine phosphate,
200
M MTX, prepare fresh.
11. 100× inhibitor mix (
see
Subheading
2.4
).
12. 100× valinomycin (
see
Subheading
2.4
).
13. 0.1 M PMSF (
see
Note 2
).
14. 2.5 mg/ml Proteinase K (PK,
see
Subheading
2.4
).
ʼ
g/ml creatine kinase, 5
ʼ
Miscellaneous: Buffers, solutions, and equipment for SDS-
PAGE, Western Blot, and Immunodecoration.
3
Methods
All centrifugation steps are to be carried out at 4 °C, unless stated
otherwise.
3.1 Preparation
of Functional
Mitochondrial
from Yeast Cells
1. Measure OD 660 nm of yeast culture: harvest ideally at
1.5-2.5 OD/ml.
2. Determine and note (!) weight of centrifugation bottles (i.e.,
500 ml bottles for Beckman JA-10 rotor). Pellet cells for 5 min
at 1,600 ×
g
and RT. Discard medium and resuspend cell pellet
in monodest. water. Fill up to 3/4 of total volume.
3. Centrifuge cell suspension again for 5 min at 1,600 ×
g
and RT.
Remove supernatant completely and etermine wet weight of
pellet.
