Biology Reference
In-Depth Information
Microdomains
Prepare stack on a semidry transfer unit as follows: thick blot paper, thin blot
paper, PVDF membrane, gel, thin blot paper, thick blot paper. Ensure there are no
bubbles between any of the layers to avoid poor transfer. Transfer for 42 min,
16 V, 0.26 limit (or 0.52 limit for two gels).
Proceed with immunoblotting using specific antibodies.
Note
: Some antibodies
are able to detect proteins in their native conformation. An alternative is to
denature the gel prior to membrane transfer by placing it in SDS containing 1
sample buffer (refer to
Section 2.1.2
) on a shaker for 30 min, boil briefly in a
microwave (e.g., 10 s), and place on a shaker for an additional 15 min. This may
allow binding by antibodies that require the protein to be denatured.
22.4.2.4
2D blue-native polyacrylamide gel electrophoresis (2D BN-PAGE)
For 2D gel electrophoresis, cut the gel slice of interest from the 1D blue-native
gel.
Place the gel slice in denaturing SDS-containing 1
sample buffer on a shaker
for 30 min, boil briefly in a microwave (e.g., 10 s), and place on a shaker for an
additional 15 min.
Prepare SDS-containing 10% polyacrylamide gels using a comb designed to hold
a gel slice and a separate well to load the molecular weight ladder.
Proceed with polyacrylamide gel electrophoresis as normal, then transfer to a
PVDF membrane, and blot with the antibody of interest.
22.4.2.5
Silver staining
For silver staining of 1D or 2D blue-native gels, the Silver Stain Plus Kit (Bio-
Rad) is recommended.
22.5
DISCUSSION
Identification of multimolecular complexes within the biochemical microenviron-
ment in which they function is a first step towards characterizing their functional role
and targeting them for therapeutic purposes. The biophysical environment in which
assembly of the complexes takes place may in itself not only define critical stages for
their formation but also focus the search for molecular interactions. We have dis-
cussed here several methods to isolate specialized membrane microdomains from
various membranes of the cell, including the plasma membrane and mitochondrial
membranes, and the analysis of multimolecular complexes and supercomplexes in
cardiolipin-enriched microdomains in the inner mitochondrial membrane.
A few aspects regarding the outlined protocols deserve further consideration. As
reported by others, in our hands, mitochondrial respiratory supercomplex isolation is
best achieved using the detergent digitonin since it is sufficiently mild to preserve the
supramolecular interactions of multichain protein complexes (
Acin-Perez et al.,
2008
). Some supercomplexes (and all individual complexes) can be extracted using
other detergents, namely, Triton X-100, NP-40 (Igepal CA-630), Tween-20, and
