Two-dimensional gel electrophoresis
(2-D electrophoresis) is a powerful and widely used method for the
analysis of complex protein mixtures extracted from cells, tissues, or
other biological samples. This technique separate proteins in two steps,
according to two independent properties: the first-dimension is
isoelectric focusing (IEF), which separates proteins according to their
isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), which separates proteins according to their
molecular weights (MW). In this way, complex mixtures consisted of
thousands of different proteins can be resolved and the relative amount
of each protein can be determined.
The procedure involves placing the
sample in gel with a pH gradient, and applying a potential difference
across it. In the electrical field, the protein migrates a long the pH
gradient, until it carries no overall charge. This location of the
protein in the gel constitutes the apparent pI of the protein.
There are two alternatives methods to
create the pH gradient - carrier ampholites and
immobilized pH gradient (IPG) gels. In the Maiman Institute for Proteome Research the IEF is performed with
commercial IPGs for highly reproducible
The IEF is the most critical step of
the 2-D electrophoresis process. The proteins must be solubilize without charged detergents, usually
in high concentrated urea solution, reducing agents and chaotrophs. To obtain high quality data it is
essential to achieve low ionic strength conditions before the IEF it
self. Since different types of samples differ in their ion content, it is
necessary to adjust the IEF buffer and the electrical profile to each
type of sample.
The separation in the second
dimension by molecular size is performed in slab SDS- PAGE. Twelve
parallel gels can be separated in a fixed temperature to minimize the
separation variations between individual gels.