Biology Reference
In-Depth Information
Cardiolipin-Enriched Mitochondrial Membrane Microdomain
Isolation
Place in an ultracentrifuge rotor (i.e., SW40, Beckman) and spin at 28,000
g
for
30 min in an Optima XL-90 ultracentrifuge (Beckman) or similar.
Carefully remove and discard all fluid phase.
Resuspend the pellet in 1 mL of lysis buffer.
Transfer into microfuge tube and keep on ice.
Wash three times with 1 mL of lysis buffer (spin in tabletop microcentrifuge at
maximum speed (e.g., 20,000
g
) for 5 s).
The pellet contains purified plasma membrane.
To solubilize proteins for Western blotting:
Add small amount of lysis buffer (i.e., 500
L) containing 1.0% SDS.
Sonicate (several short bursts, keep on ice as much as possible).
Boil for 5 min.
Pellet on tabletop centrifuge at 20,000
m
g
for 10 min at 4
C.
The supernatant now contains solubilized proteins from the plasma
membranes. Transfer the supernatant to a new tube, add 4
sample buffer,
then boil, and use for Western blotting.
Discard the silica pellet.
22.3
CARDIOLIPIN-ENRICHED MITOCHONDRIAL MEMBRANE
MICRODOMAIN ISOLATION
The following protocol is designed to isolate cardiolipin-enriched microdomains
from mitochondrial membranes. This protocol has been adapted from the method
introduced by
Ciarlo et al. (2010)
.
22.3.1
Materials
22.3.1.1
Reagents
HEPES
NaCl
Triton X-100
Aprotinin
Tris-HCl, pH 8.8
SDS
EDTA
Tris, pH 7.5
Sucrose
ddH
2
O
22.3.1.2
Buffers
Isolation buffer
250 mM sucrose
