DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 9

than 0.1 pH units. The “gel” component of IEF gels is usually polyacry-
lamide, although agarose can be used. The separation is achieved by
introduction of “ampholytes”, which are synthetic compounds of differing
pHs. When subjected to an electric current, they form a pH gradient. Pro-
teins will migrate toward that position at which they have no net charge
(the “zwitterions” form), which reflects their isoelectric point.
IEF is an especially useful system for analyzing the microheterogen-
eity of proteins, such as revealing differences in glycosylation status,
or identifying different phospho-isoforms. Frequently, these differences
are not revealed on SDS-PAGE but are readily evident on IEF.
Two-dimensional (2D) gel electrophoresis
2D gel electrophoresis is a way to couple different gel systems with
different resolving powers to dramatically improve separations and res-
olution of complex mixtures of proteins. 2D gel electrophoresis is an
incredibly useful analytic pool, and provides a foundation for what is
now referred to as “proteomics”. The two most common types of 2D gel
combine the power of IEF with SDS-PAGE, or combines non-reducing
with reducing SDS-PAGE. In the first combination, proteins are sep-
arated in tubes by IEF, and the first gel is then treated so that the
proteins can migrate out of the gel. This gel is placed at the top of
the second gel and subjected to the next separation technique (SDS-
PAGE). This allows for fine detail mapping of protein composition of
cells (or subcellular organelles). If the proteins have come from cells
that have been metabolically labeled with radioactive precursors, they
can be directly visualized on film. Alternatively, 2D gels can be silver
stained to reveal differences in protein composition of cells (or subcel-
lular organelles) at different stages of development, or upon treatment
with a drug, or even to monitor changes in protein expression during an
infection with bacteria or viruses. This may involve what is euphemisti-
cally called the stare and compare approach, where long hours are
spent looking for differences between two gels. More recently, computer
programs that help overlay 2 different gels and identify differences be-
tween them have been developed to help expedite analysis. The identity
of the proteins that change in expression can then be identified by can-
didate antibodies (by western blotting) or by sequencing using mass
spectroscopy.
Two-dimensional non-reducing/reducing gels are processed similarly.
The protein mixtures (for example, extracts from cells that have been
metabolically labeled) are fractionated first under non-reducing and non-
denaturing conditions. The gel is turned on its side and the proteins
resolved in the first dimension are then denatured and resolved in the