Biology Reference
In-Depth Information
DDM (
Acin-Perez et al., 2008
). As a control to measure the total amount of individ-
ual complexes, we recommend treating the samples of interest with DDM since all
individual complexes are detectable by Western blotting following this treatment
with no detectable proportion associated with supercomplexes. Meanwhile, the de-
tergents Brij-96V, cholate, Empigen BB, perfluorooctanoic acid, and CHAPS are
largely unable to extract any individual complexes or supercomplexes (
Acin-
Perez et al., 2008
).
It is also important to keep in mind that the detection of an array of molecules
within the fraction of detergent-insoluble microdomains of a given compartment
does not necessarily correlate with colocalization of these molecules within the same
microdomains. This is due to the heterogeneity of lipid rafts, determined by many
factors including ectodomain interactions between surface receptors (
Pike, 2004;
Wang, Gunning, Kelley, & Ratnam, 2002
).
Finally, we acknowledge that there is an ongoing debate about the biological sig-
nificance of the biochemical preparation of detergent-insoluble microdomains
and the multimolecular complexes that are isolated within these compartments.
There is still controversy on whether these domains do actually exist as discrete en-
tities
in vivo
or rather result from the biochemical manipulation of the cells and their
membranes. This debate is apparent when discussing the existence and biological
significance of mitochondrial respiratory supercomplexes. Assessment of the differ-
ent sides of this argument goes beyond the scope of this methodological paper.
SUMMARY
It is becoming increasingly apparent that there are specialized membrane microdo-
mains in many biological membranes, including the plasma and mitochondrial mem-
branes, and that these microdomains are heterogeneous with varying compositions.
The support for the existence of these microdomains in a natural setting and their
formation, independently of biochemical manipulation or as an experimental arti-
fact, is still under debate. However, through the use of different approaches, includ-
ing those outlined in this chapter, it is becoming increasingly clear that these
specialized membrane microdomains are biologically relevant in distinct ways
depending on their location and composition. It is hoped that the techniques detailed
here will be helpful to explore novel models to test and verify the functional corre-
lates that will support their biological significance.
Acknowledgments
We thank Dr. Eric Shoubridge (McGill University, Montr´al, QC) for helpful comments and
discussions in the development of protocols for mitochondrial isolation and 2D BN-PAGE.
Work at the Madrenas laboratory is funded by the CIHR. J.M. holds a Tier I Canada Research
Chair in Human Immunology.
