Biology Reference
In-Depth Information
Collect the supernatants containing Triton X-100-soluble material. Centrifuge
the pellets a second time (20,000
g
for 30 sec at 4
C) to remove the remaining
soluble material.
Solubilize pellets in 100
L solubilization buffer.
Shear DNA by passage through a 22-gauge needle.
Analyze both Triton X-100-soluble and Triton X-100-insoluble materials by
Western blot. Load the fraction samples by volume.
m
22.4
MITOCHONDRIAL SUPERCOMPLEX IDENTIFICATION
BY BLUE-NATIVE GEL ELECTROPHORESIS
Blue-native gel electrophoresis was first applied to digitonin-solubilized mitochon-
drial membranes by
Schagger and Pfeiffer (2000)
leading to the identification of mi-
tochondrial respiratory supercomplexes. Blue-native refers to the fact that the
separated protein complexes are not denatured but rather retain enzymatic activity
while their electrophoretic separation relies on binding of the dye Serva Blue G
to the protein. Treatment of samples with the mild detergent digitonin allows
for many protein-protein interactions to remain intact, allowing for analysis of
supramolecular associations of the respiratory complexes and other proteins.
Blue-native samples are run on a first-dimension nondenaturing polyacrylamide
gel to separate protein complexes based on size. Analysis of the components of
each multiprotein complex from the first-dimension gel can be achieved by cutting
the appropriate gel slice and running it on a denaturing second-dimension poly-
acrylamide gel followed by Western blotting. Silver staining may also be performed
on first- or second-dimension gels to visualize multiprotein complexes or their
components, respectively. This technique can be used to gather a considerable
amount of information on multiprotein complexes and mitochondrial respiratory
supercomplexes in particular. For this reason, blue-native gel electrophoresis
is now one of the most commonly used techniques for studying mitochondrial
respiratory supercomplexes. The following protocol for the isolation of mito-
chondrial respiratory supercomplexes by blue-native gel electrophoresis has been
adapted from
Schagger and von Jagow (1991)
and
Sasarman, Antonicka, and
Shoubridge (2008)
.
22.4.1
Materials
22.4.1.1
Reagents
Aminocaproic acid
Bis-tris
Tricine
Serva Blue G
Acrylamide/bisacrylamide
EDTA