soelectric focusing (IEF) is an
electrophoretic separation
based on the isoelectric points
of proteins. The pI is the point
at which the protein has an
overall net charge of zero.
Differences of only a few hun-
dredths of a pH-unit in isoelec-
tric points are sufficient to resolve proteins from
each other. IEF is used as an alternative elec-
trophoresis format complementing the widely
used SDS-PAGE electrophoresis, which is based
on size of proteins. The combination of both
principles is applied in 2D-electrophoresis as out-
lined below (see page 2). This technique has be-
come crucial for the development of Proteomics.
In many applications, closely related proteins
have to be separated. Examples are the differenti-
ation of protein isoforms or enantiomeres. Such
problems have been successfully solved by iso-
electric focusing (IEF) [1]
In contrast to other electrophoretic techniques,
pH is not kept constant throughout the whole
system. Instead, the sample components migrate
electrophoretically in a stationary pH-gradient.
Proteins will migrate until they reach the pH-
point in the gradient at which the charge of the
protein equals zero (pH = pI) The protein is said
to focus at this point. This focusing also results in
a concentration of individual proteins (Figure 1)