Protein Protocols Protein Protocols

288 Dunn
variations in silver staining methodology (6,8). This group of procedures is generally
accepted to be approx 100 times more sensitive than methods using CBB R-250, being
able to detect low nanogram amounts of protein per gel band or spot. Although silver
staining has become the most widely used technique for high sensitivity visualization
of gel separated proteins (see Chapter 33), its variable binding characteristics toward
many proteins and its relatively restricted dynamic range limit the accuracy and reliability
of quantitation. Moreover, saturated protein bands and spots tend to be negatively
stained making their quantitation impossible. Standard methods of silver staining
are also often not compatible with chemical characterization by mass spectrometry as
the glutaraldehyde and formaldehyde present results in alkylation of α- and ε-amino
groups of proteins. To overcome this problem, silver staining protocols compatible
with mass spectrometry in which glutaraldehyde is omitted have been developed (9,10)
but these suffer from a decrease in sensitivity of staining and a tendency to a higher
background.
Many of the problems associated with silver staining can be overcome using detection
methods based on the use of fluorescent compounds. This group of methods are
highly sensitive and generally exhibit excellent linearity and a high dynamic range,
making it possible to achieve good quantitative analysis, particularly if a suitable
imaging device is used. In addition, these methods should in general have good compatibility
with protein characterization methods. Two approaches can be used, the first
being to couple the proteins with a fluorescently labeled compound prior to electrophoresis.
Examples of such compounds are dansyl chloride (11), fluorescamine
(4-phenyl-[furan-2(3H),1-phthalan]-3,3'-dione) (12), o-pththaldialdehyde (OPA) (13), and
MDPF (2-methoxy-2,4-diphenyl-3([2H])-furanone) (MDPF) (14). The latter reagent
has a reported sensitivity of 1 ng protein/band and is linear over the range 1–500 ng of
protein/band.
The main disadvantage of preelectrophoretic staining procedures is that they can
cause protein charge modifications; for example, fluorescamine converts an amino
group to a carboxyl group when it reacts with proteins. Such modifications usually
do not compromise sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) unless a large number of additional charged groups are introduced into the
protein. However, they result in altered mobility during other forms of electrophoresis,
resulting in altered separations by native PAGE, isoelectric focussing (IEF), and 2-DE.
Recently, compounds that react with cysteine or lysine residues have been described
and used successfully for 2-DE separations. The cysteine-reactive reagent
monobromobimane (15) has been used to label proteins prior to analysis by 2-DE (16).
Using a cooled CCD camera to measure fluorescence , the limit of detection was found
to be 1 pg protein/spot (17).
In an alternative approach, two amine-reactive dyes (propyl Cy3 and methyl Cy5)
have been synthesized and used to label E. coli proteins prior to electrophoresis (18).
These cyanine dyes have an inherent positive charge, which preserves the overall charge
of the proteins after dye coupling. The two dyes have sufficiently different fluorescent
spectra that they can be distinguished when they are present together. This allowed two
different protein samples, each labeled with one of the dyes, to be mixed together and
subjected to 2-DE on the same gel. This method, which has been termed difference gel