404 Part III: Muscle Foods
several drawbacks. The most significant of these
have to do with the diversity of proteins and their
expression levels. For example, hydrophobic pro-
teins do not readily dissolve in the buffers used for
isoelectrofocusing. This problem can be overcome,
though, using nonionic or zwitterionic detergents,
allowing for 2DE of membrane and membrane-
associated proteins (Babu et al. 2004, Chevallet et
al. 1998, Henningsen et al. 2002, Herbert 1999).
Vilhelmsson and Miller (2002), for example, were
able to use “membrane protein proteomics” to de-
monstrate the involvement of membrane-associated
metabolic enzymes in the osmoadaptive response of
the foodborne pathogen Staphylococcus aureus. A
2DE gel image of S. aureusmembrane-associated
gels is shown in Figure 18.4.
Similarly, resolving alkaline proteins, particularly
those with pH above 10, on 2D gels has been prob-
lematic in the past. Although the development of
highly alkaline, narrow-range IPGs (Bossi et al.
1994) allowed reproducible two-dimensional reso-
lution of alkaline proteins (Görg et al. 1997), their
representation on wide-range 2DE of complex mix-
tures such as cell extracts remained poor. Improve-
ments in resolution and representation of alkaline
proteins on wide-range gels have been made (Görg
Figure 18.3.A screenshot from the 2DE analysis program Phoretix 2-D (NonLinear Dynamics, Gateshead, Tyne and
Wear, United Kingdom) showing some steps in the analysis of a two-dimensional protein map. Variations in abun-
dance of individual proteins, as compared with a reference gel, can be observed and quantified.
Figure 18.4.A 2DE membrane proteome map from
Staphylococcus aureus,showing proteins with pH
between 3 and 10 and molecular mass about 15–100
(O. Vilhelmsson and K. Miller, unpublished).
Isoelectrofocusing was in the presence of a mixture of
pH 5–7 and pH 3–10 carrier ampholytes, and the sec-
ond dimension was in a 10% polyacrylamide slab gel
with a 4% polyacrylamide stacker.