Biology Reference
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￿ 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
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