DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 8

Reducing and non-reducing SDS-PAGE
Most SDS-PAGE gels are run under reducing conditions, meaning
that agents that reduce disulfide bonds, such as 2-mercaptoethanol
(2-ME), are incorporated into the SDS-containing loading buffers. The
presence of 2-ME serves two functions. First, it reduces the covalent
bonds that might exist between muitimeric protein complexes. Second,
it reduces covalent bonds that exist within a protein so that it achieves
a more extended structure when boiled in SDS, which improves resolu-
tion and permits more accurate estimates of its molecular mass. Under
non-reducing conditions, proteins can migrate aberrantly on SDS-PAGE
gels. However, the comparison of isolated proteins on reducing and non-
reducing SDS-PAGE (such as when coupled with immunoprecipitation
using specific antibodies) provides an easy way to determine if a pro-
tein of interest might be covalently associated with another protein within
a cell.
Native (non-denaturing) PAGE gels
Although SDS-PAGE is the most commonly used system for analyzing
proteins, it does have some drawbacks. First, because of the denaturing
activity of the SDS, it is often impossible to recover proteins in functional
form, for example to measure enzymatic activity, once they have been
fractionated on SDS-PAGE. In addition, important non-covalent protein-
protein interactions are disrupted by SDS. Native gels offer advantages
for both of these purposes. Native PAGE gels lack SDS. As a result,
proteins migrate according to their own intrinsic charge (due to charged
amino acids or post-translational modifications) as well as the sieving
properties of the gel. In other words, they migrate very differently than
on SDS-PAGE. Often, proteins can migrate as non-covalent complexes
on these gels. Native gels in tubes can be used as a first separation for
2-dimensional gel electrophoresis, where complexes are separated in
the first dimension in native gels, and then in the second by reducing
SDS-PAGE. This helps in determining the nature of stable, non-covalent
complexes (see below).
Isoelectric focusing (IEF)
Isoelectric focusing gels separate proteins according to their isoelec-
tric points (pl). The pl of a protein is defined as the pH at which it has no
net charge; below that pH, the protein has a net positive charge, while
above it the protein is negatively charged. Isoelectric focusing gels are
capable of high resolution, separating proteins with pls that differ by less