Bio-Rad EXQuest Spot Cutter - Manual (Page 11)

PDQuest User Guide
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2. Overview of Raw 2-D Gel Electrophoresis
Raw 2-D electrophoresis is a method for separating proteins and nucleic acids in a
sample into a two-dimensional pattern of spots in a gel. It combines the techniques of
isoelectric focusing (IEF) with SDS-polyacrylamide gel electrophoresis (SDS-
PAGE). Since these two separation methods rely on independent properties of
proteins—chemical and physical—this procedure can resolve complex biological
samples with a high degree of specificity and accuracy. The resolved proteins and
polypeptide fractions can be then identified by their molecular weights and charges
(as indicated by their locations in the Raw 2-D gel), as well as by their differential
expression in different samples, proximity to other spots, intensity, etc.
Raw 2-D electrophoresis involves two sequential separations of a sample in
perpendicular directions. The IEF dimension is run first, in tube gels or on
immobilized pH gradient (IPG) strips. After focusing, the tube gel or strip is placed
on top of an SDS-PAGE slab gel and electrophoresed. This technique can resolve
thousands of protein spots in a single sample; these proteins can then be visualized by
metabolic radiolabeling or a variety of staining methods.
Proteomics Applications
Proteomics is the study of protein expression and regulation in cells, tissues, and
entire organisms. Several thousand proteins are expressed at any given moment in an
organism; at the cellular level, dozens of proteins may be expressed and regulated in
fractions of a second.
Two-dimensional gel electrophoresis is a a cornerstone in the study of how proteins
are expressed, regulated, and modified throughout living systems. Developed almost
a quarter of a century ago, Raw 2-D gel technology remains one of the most powerful
techniques for resolving complex mixtures of proteins. The technology has improved
significantly over the past several years, with the advent of IPG strips and simplified
gel running techniques, large-format and cyber gels that allow for greater pH range
and specificity, and new stains and staining techniques. In addition, mass
spectrometry now allows for peptide mass fingerprinting of very small amounts of
protein isolated from gels.
Using a combination of these techniques, pharmaceutical companies can now use
Raw 2-D technology for high-throughput screening of drug compound candidates
using protein targets; research laboratories can study large-scale changes in protein