Biology Reference
In-Depth Information
Microdomains
22.4 Mitochondrial Supercomplex Identification by Blue-Native Gel Electrophoresis...423
22.4.1 Materials ................................................................................... 423
22.4.1.1 Reagents ........................................................................... 423
22.4.1.2 Equipment......................................................................... 424
22.4.1.3 Buffers and Solutions......................................................... 424
22.4.2 Method...................................................................................... 425
22.4.2.1 Sample Preparation ........................................................... 425
22.4.2.2 Performing Blue-Native Gel Electrophoresis........................ 426
22.4.2.3 Analysis by Western Blotting .............................................. 426
22.4.2.4 2D Blue-Native Polyacrylamide Gel Electrophoresis
(2D BN-PAGE) ................................................................................. 428
22.4.2.5 Silver Staining.................................................................... 428
22.5 Discussion......................................................................................................428
Summary ................................................................................................................ 429
Acknowledgments ................................................................................................... 429
References ............................................................................................................. 430
Abstract
Cellular membranes contain specialized microdomains that play important roles in a
wide range of cellular processes. These microdomains can be found in the plasma
membrane and other membranes within the cell. Initially labeled lipid rafts and
defined as being resistant to extraction by nonionic detergents and enriched in cho-
lesterol and glycosphingolipids, we now understand that these membrane microdo-
mains are very dynamic and heterogeneous membrane structures whose composition
and function can vary widely depending on their cellular location. Indeed, though
they are classically associated with the plasma membrane and have been shown
to facilitate a wide variety of processes, including signal transduction and membrane
trafficking, specialized membrane microdomains have also been identified in other
membranes including those in the mitochondria. These mitochondrial membrane
microdomains are enriched in cardiolipin, the signature phospholipid of the mito-
chondria, and may have important implications in metabolism by facilitating optimal
assembly and function of the mitochondrial respiratory chain. Furthermore, isolation
of multimolecular complexes while retaining their supramolecular interactions
has been critical to the study of mitochondrial respiratory supercomplexes. Here,
we discuss methods to isolate various membrane microdomains, including
detergent-insoluble glycosphingolipid microdomains, mitochondrial cardiolipin-
enriched microdomains, and blue-native gel electrophoresis of mitochondrial
membranes.
