Protein Protocols Protein Protocols

Fluorescent Staining of Gels 287
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Detection of Proteins in Polyacrylamide Gels
by Fluorescent Staining
Michael J. Dunn
1. Introduction
Techniques of polyacrylamide gel electrophoresis are often the method of choice for
the analysis of patterns of gene expression in a wide variety of complex systems. In
particular, two-dimensional polyacrylamide gel electrophoresis (2-DE) is the core technology
for separating complex protein mixtures in the majority of proteome projects
(1). This is due to its unrivalled power to separate simultaneously thousands of proteins
and the availability of sophisticated computer software for the qualitative and quantitative
analysis of differential patterns of protein expression (2). Once this analysis has
indicated proteins of interest, it is now relatively straightforward to directly identify
and characterize proteins from polyacrylamide gels using highly sensitive microchemical
methods (3), particularly those based on mass spectrometry (4). This has resulted in
gel electrophoresis becoming one of the most important methods of protein purification
for subsequent identification and characterization.
The successful use of polyacrylamide gel electrophoresis as an analytical tool for
investigating protein expression and as a micropreparative procedure for protein identification
and characterization depends on the availability of detection methods that are
both quantitative and sensitive. Coomassie Brilliant Blue R-250 (CBB R-250) has been
used for many years as a general protein stain following gel electrophoresis. Its
restricted dynamic range limits its suitability for accurate quantitative analysis,
although it is compatible with most methods of chemical characterization. However,
CBB R-250 has a limited sensitivity being capable of detecting microgram to submicrogram
amounts of protein. The development of colloidal CBB staining using CBB
G-250 (5) provides increased sensitivity at approx 10 ng of protein. However, the trend
toward the use of thinner gels and the need to detect small amount of protein within
single bands or spots resolved by one- or two-dimensional electrophoresis have necessitated
the development of more sensitive detection methods (6).
Silver staining for the detection of proteins following gel electrophoresis was first
reported in 1979 by Switzer et al. (7), resulting in a major increase in the sensitivity of
protein detection. More than 100 publications have subsequently appeared describing
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
287