BLBS102-c22 BLBS102-Simpson March 21, 2012 13:41 Trim: 276mm X 219mm Printer Name: Yet to Come
408 Part 3: Meat, Poultry and Seafoods67 kDa43 kDa30 kDa21 kDa14 kDa(^4) pI^7
Figure 22.2.A two-dimensional electrophoresis protein map of
rainbow trout (Oncorhynchus mykiss) liver proteins with pI between
4 and 7 and molecular mass about 10–100 (S. Martin, unpublished).
The proteins are separated according to their pI in the horizontal
dimension and according to their mass in the vertical dimension.
Isoelectro focussing was by pH 4–7 immobilized pH gradient (IPG)
strip and the second dimension was in a 10–15% gradient
polyacrylamide slab gel.
abundance is taken into consideration by running gels from a
sufficient number of samples and performing the appropriate
statistics. Pooling samples may also be an option, depending on
the type of experiment.
Basic 2DE Methods Overview
O’Farrell’s original 2DE method first applies a process called
isoelectric focusing (IEF), where an electric field is applied to
a tube gel on which the protein sample and carrier ampholytes
have been deposited. This separates the proteins according to
their molecular charge. The tube gel is then transferred onto
a polyacrylamide slab gel and the isoelectrically focused pro-
teins are further separated according to their molecular mass
by conventional sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE), yielding a two-dimensional map
(Fig. 22.2) rather than the familiar banding pattern observed in
one-dimensional SDS-PAGE. The map can be visualized and
individual proteins quantified by radiolabeling or by using any
of a host of protein dyes and stains, such as Coomassie blue,
silver stains, or fluorescent dyes. By comparing the abundance
of individual proteins on a number of gels (Fig. 22.3), upregu-
lation or downregulation of these proteins can be inferred. Al-
though a number of refinements have been made to 2DE since
O’Farrell’s paper, most notably, the introduction of immobilized
pH gradients (IPGs) for IEF (Gorg et al. 1988), the procedure ̈
Figure 22.3.A screenshot from the two-dimensional electrophoresis analysis program Phoretix 2-D (NonLinear Dynamics, Gateshead, Tyne
& Wear, UK) showing some steps in the analysis of a two-dimensional protein map. Variations in abundance of individual proteins, as
compared with a reference gel, can be observed and quantified.