A Thousand Points of Light The Application of Fluo

Electrophoresis 2000, 21, 11231144
1123
Review
Wayne F. Patton
Molecular Probes, Inc.,
Eugene, OR, USA
A thousand points of light: The application of

fluorescence detection technologies to twodimensional gel electrophoresis and proteomics
As proteomics evolves into a high-throughput technology for the study of global protein
regulation, new demands are continually being placed upon protein visualization and
quantitation methods. Chief among these are increased detection sensitivity, broad linear dynamic range and compatibility with modern methods of microchemical analyses.
The limitations of conventional protein staining techniques are increasingly being
encountered as high sensitivity electrophoresis methods are interfaced with automated
gel stainers, image analysis workstations, robotic spot excision instruments, protein
digestion work stations, and mass spectrometers. Three approaches to fluorescence
detection of proteins in two-dimensional (2-D) gels are currently practiced: covalent
derivatization of proteins with fluorophores, intercalation of fluorophores into the
sodium dodecyl sulfate (SDS) micelle, and direct electrostatic interaction with proteins
by a Coomassie Brilliant Blue-type mechanism. This review discusses problems
encountered in the analysis of proteins visualized with conventional stains and
addresses advances in fluorescence protein detection, including immunoblotting, as
well as the use of charge-coupled device (CCD) camera-based and laser-scannerbased image acquisition devices in proteomics.
Keywords: Protein stain / Two-dimensional gel electrophoresis / Mass spectrometry / Fluores-
Contents
1
Introduction . . . . . . . . . . . . . . . . . . . . . . .
1.1 Brief summary of nonfluorescent protein
visualization techniques . . . . . . . . . . . . . . .
1.2 Shortcomings of standard protein detection
methods: sensitivity and linearity . . . . . . . . .
2
Covalent labeling of proteins with fluorophores
2.1 Monobromobimane . . . . . . . . . . . . . . . . . .
2.2 MDPF . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3 Propyl Cy3/methyl Cy5. . . . . . . . . . . . . . . .
2.4 Merits of derivatizing amino versus sulfhydryl
groups . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5 Covalent derivatization: caught between a rock
and a hard place . . . . . . . . . . . . . . . . . . . .
1123
1124
1125
1126
1127
1128
1128
1129
1131
Correspondence: Wayne F. Patton, PhD, Bioanalytical Assay

Development Group, Molecular Probes, Inc., 4949 Pitchford Avenue, Eugene, OR, 97402, USA
E-mail: waynepatton@probes.com
Fax: +541-344-6504
Abbreviations: LED, light emittive diode; MDPF, 2-methoxy2,4-diphenyl-3(2H)furanone; Nd-YAG, neodymium-yttrium-aluminum garnet
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000
EL 3838
3
3.1
3.2
3.3
3.4
4
4.1
4.2
4.3
5
5.1
5.2
5.3
6
7
Noncovalent fluorescent staining of proteins .

Nile Red dye. . . . . . . . . . . . . . . . . . . . . . .
SYPRO Orange, Red and Tangerine dyes . .
SYPRO Rose and SYPRO Ruby dyes . . . . .
Whats old is new again . . . . . . . . . . . . . . .
Luminescence immunodetection on
electroblots . . . . . . . . . . . . . . . . . . . . . . .
Dominance of chemiluminescence detection .
Direct fluorescence detection . . . . . . . . . . .
Fluorogenic alkaline phosphatase and
horseradish peroxidase substrates. . . . . . . .
Fluorescence imaging devices . . . . . . . . . .
Overview of fluorescence detection
instrumentation . . . . . . . . . . . . . . . . . . . . .
CCD camera-based systems . . . . . . . . . . .
Laser scanner systems . . . . . . . . . . . . . . .
Conclusions and perspectives . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
1132
1132
1133
1134
1135
1136
1136
1136
1137
1138
1138
1138
1139
1141
1141
1 Introduction
In Dr. Bonnie Dunbars definitive 372-page monograph
entitled Two-dimensional Electrophoresis and Immunological Techniques, published in 1987, fluorescence
0173-0835/00/0606-1123 $17.50+.50/0
Proteomics and 2-DE
cence / Proteomics / Review
1124
W. F. Patton
detection methods for protein visualization were discussed in two sentences of the last section of the protein
detection chapter, under the less-than-auspicious heading Miscellaneous Protein-Staining Methods [1]. The
use of fluorescence-based approaches for protein detection has become more commonplace in the intervening
years, yet the majority of researchers still rely more extensively upon radiolabeling, Coomassie Brilliant Blue (CBB)
staining and silver staining. In the past, the use of fluorescent protein stains has been considered by some to be
inconvenient since UV illumination is usually necessary
for visualization and direct quantitation requires fairly
sophisticated instrumentation. The measurement of light
emission is intrinsically more sensitive than measurement
of light absorbance, however, as absorption is limited by
the molar extinction coefficient of the colored complex [2].
Thus, fluorescent protein stains often provide greater sensitivity and broader linear dynamic responses when
compared to their colorimetric counterparts. The widespread adoption of ethidium bromide, SYBR Green and
SYBR Gold stains for fluorescence detection of nucleic
acids has bolstered acceptance of fluorescent stains as
tools for the routine analysis of proteins.
Since the 1980s, numerous bioanalytical assays have

gravitated away from the use of radioisotopes towards
implementation of nonisotopic detection systems. The
hazards of working with radioactivity, limited shelf-life, disposal costs, and problems with handling mixed waste
have contributed to this fundamental change. By comparison, fluorophores are easy to handle, have long shelf
lives, and are readily disposed of by incineration so that
there are minimal storage and disposal issues. Recent
technological developments in fluorescence methodologies and instrumentation have allowed for the evolution of
new assays with improved sensitivity and specificity, permitting the detection and quantification of analytes that
would otherwise be difficult to measure by any other
approach. These improvements have launched fluorescence into the midst of a wide range of mainstream applications relevant to research and development, the
pharmaceutical and clinical diagnostics industries. Fluorescence detection of proteins in polyacrylamide gels is
still an emerging technology, but now that proteomics has
caught the attention of large pharmaceutical companies
wishing to add the approach to their repertoire for largescale screening programs, substantial investments are
being made to facilitate implementation of the technology.
This includes the development of integrated work environments consisting of automated gel stainers, image analysis workstations, robotic spot excision instruments, protein digestion work stations, and mass spectrometers
(reviewed in [3]).
1.1 Brief summary of nonfluorescent protein

visualization techniques
Staining in aqueous solutions containing methanol, acetic
acid, and CBB R-250, followed by destaining in a comparable solution lacking dye endures as a ubiquitous method
for visualization of proteins separated by polyacrylamide
gel electrophoresis [4, 5].
However, stoichiometric binding of dye to proteins is difficult to achieve because proteins are decolorized to differing extents during the destaining procedure. Thus, rigorous quantitation of CBB R-250-stained proteins is somewhat problematic. CBB R-250 dye is capable of detecting mg to sub-mg amounts of protein [68]. The development of colloidal CBB staining using CBB G-250 (dimethylated CBB R-250) for background-free detection of
proteins in polyacrylamide gels offered substantial improvements over previous CBB staining procedures [9
11]. The limit of protein detection for colloidal CBB stain
is approximately 810 ng of protein and since the destaining step is avoided, quantitation is more reliable, though
the linear dynamic range of the dye is relatively small [8].
Studies of protein modifications by mass spectrometry
indicate that staining proteins in colloidal CBB dye solutions containing trichloroacetic acid and alcohol leads to
irreversible, acid-catalyzed esterification of glutamic and
aspartic acid side chain carboxyl groups [12]. This can
complicate interpretation of peptide mapping data from
mass spectrometry. However, our group finds that stain
formulations using dilute phosphoric acid with methanol
instead of trichloroacetic acid do not suffer from this problem.
While Amido Black was one of the earliest organic dyes
used to visualize proteins after electrophoresis, it is now
principally used for medium sensitivity, colorimetric detection of electroblotted proteins of PVDF and nitrocellulose
membranes [13, 14]. Both Amido Black and CBB dyes
are suitable for detection of proteins electroblotted to
PVDF membranes. Amido Black is also fully capable of
staining proteins on nitrocellulose membranes, while a
modified approach for low background CBB staining of
proteins on nitrocellulose membranes has been developed as well [15]. Amido Black and CBB staining of electroblotted proteins on membranes permits subsequent
downstream microchemical characterization but often
interferes with immunoblotting and photographic documentation [16, 17]. This problem may be circumvented by
destaining the proteins prior to immunodetection or by
running duplicate blots, one for total protein detection and
one for immunodetection [16, 18, 19]. Direct correlation
between the total protein profile and bands detected with
antibodies is sometimes difficult using such approaches.

Kerenyi and Gallyas [20] first devised a general silver
staining method for the detection of proteins separated by
electrophoresis in agarose gels, with detection sensitivity
reported to be roughly 1020 times better than Amido
Black stain. Seven years later, a method for silver staining
of proteins in polyacrylamide gels was reported that was
at least 100 times more sensitive than CBB staining [21].
This second method, in combination with a variety of others developed shortly thereafter, quickly established silver
staining as the preeminent protein detection technique in
biological research laboratories worldwide. For the first
time, nonradioactive detection of proteins in the ng range
instead of the mg range was possible.
Typically, two kinds of silver staining are used for the
detection of proteins in polyacrylamide gels: acidic silver
nitrate and alkaline silver diamine methods [22]. Both approaches require an oxidation step followed by reduction
to metallic silver [23]. The acidic silver nitrate methods
are based upon photographic procedures and rely upon
gel impregnation with silver ions at acidic pH, followed by
reduction to elemental metallic silver at alkaline pH using
formaldehyde [7, 23]. The alkaline silver diamine methods
are based upon histological procedures that use ammonium hydroxide to form soluble silver diamine complexes
followed by visualization through reduction of free silver
ions with formaldehyde in an acidified developer [7, 23].
The acidic silver nitrate procedures appear to stain basic
proteins with slightly lower sensitivity and acidic proteins
with higher sensitivity than alkaline silver diamine methods [22, 24]. Gels utilizing the Tris, Tricine or Bicine buffer
chemistry, such as certain commercially available, long
shelf life, precast gels and gels routinely used for the
separation of low molecular weight proteins, are poorly
stained using the alkaline silver diamine methods. Proteins separated using immobilized pH gradient gels are
also poorly stained using alkaline silver diamine methods
[24]. Due to the volatile nature of ammonia, the alkaline
silver diamine methods are more susceptible to run-to-run
variability than the acidic silver nitrate methods [22].
Numerous reverse stain methods for the visualization of
proteins in SDS-polyacrylamide gels have been described
over the years, but the three methods most extensively
used are the potassium chloride, copper chloride and zinc
chloride procedures [2528]. Reverse stains produce a
semiopaque background on the gel surface, while proteins are detected as transparent zones when gels are
viewed on a black background or with proper back illumination [7]. Staining is quite rapid, usually requiring 515
min to complete, and the biological activity of proteins is
often preserved. Protein elution may be achieved after
chelation of the metal ions with agents such as ethylenediaminetetraacetic acid (EDTA) [26, 29]. Reverse stains
Fluorescence detection and imaging
1125
are suitable for visualization of proteins, their passive elution from gels, and analysis by mass spectrometry [30].
Needless to say, the stains can not be used to detect proteins on electroblot membranes.
Finally, a number of colloidal dispersions have found
application for protein detection on electroblots. India ink
was the first successful colloidal dispersion stain used in
this capacity [31]. The principal advantage of the staining
procedure is that it is simple to perform and inexpensive.
However, it has been noted that India ink displays relatively high protein-to-protein staining variability compared
with most other stains in common use [14, 17, 3234].
Shortly after the development of India ink as a stain for
electroblotted proteins, other particle dispersions were
perfected for the same application [32, 35, 36]. The most
widely used of these has been colloidal gold staining.
Methods for using colloidal gold staining in conjunction
with colorimetric or chemiluminescent immunoblotting
procedures have been reported [3739].
1.2 Shortcomings of standard protein detection

methods: sensitivity and linearity
Renewed interest in protein detection technologies has
arisen from a need to combine high detection sensitivity
with broad quantitation capabilities. Though the primary
aim of proteomics studies is to define biological processes at the protein level in both qualitative and quantitative terms, it is often overlooked by practitioners of the art
that the tools for quantitatively evaluating protein expression are largely insufficient to the task. An intrinsic
advantage of fluorescence technology is that the detection of the primary signal provides a linear response with
respect to the amount of protein over a much wider range
than is found for the nonfluorescent alternatives (Fig. 1).
The dynamic range of quantitation obtained with fluorescent electrophoresis stains matches the dynamic range of
gene expression levels measured in yeast, as determined
by serial analysis of gene expression (SAGE; 0.3200
transcript copies per cell) [40]. Among the nonfluorescent
detection technologies, only radiolabeling provides comparable performances.
Increase in the sensitivity of sequencing techniques and
mass spectrometry have transformed the role of protein
stains from simple visualization reagents to key components of integrated protein characterization platforms [3].
Using mass spectrometry, the determination of peptide
masses at the middle fmole level is practicable [41]. One
hundred fmole of a 15200 kDa protein corresponds to
1.520 ng of material. Thus, current methods of proteome
analysis require protein detection technologies that routinely deliver very low ng levels of sensitivity. Among the
1126
W. F. Patton
nonfluorescent staining methods, only colloidal gold, silver, and zinc-imidazole reverse staining techniques achieve this requisite level of sensitivity.
Though colloidal gold staining offers excellent detection
sensitivity for electroblotted proteins, the stain displays a
very restricted linear dynamic range for protein quantitation [34, 42, 43]. In addition, the method requires pretreatment of the membrane with a blocking agent such as
Tween-20, followed by incubation in detergent-stabilized
colloidal gold solution for 118 h [35]. This detergent
treatment partially removes proteins from the transfer
membrane, resulting in lower overall yields of material for
later characterization [44, 45]. Baking membranes at
100oC or fixing proteins with agents such as glutaraldehyde or potassium hydroxide reduces protein losses arising from detergent extraction but often renders the proteins unusable for other applications [44]. Nonionic
detergents also interfere with the collection of high quality
spectra by matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) and electrospray ionization
(ESI) mass spectrometry [46, 47]. As a case in point,
Tween-20 generates polymer background with a massto-charge ratio range of 5001200, severely suppresses
signal, and forms adducts with proteins in ESI-MS [47]. In
fact, this author is not aware of any literature reports demonstrating the successful use of colloidal gold staining in
conjunction with mass spectrometry of peptides. Moreover, colloidal gold staining consistently interferes with
protein sequence analysis by Edman degradation as
established by poor initial and repetitive sequencing
yields [48].
The numerous solution changes and carefully timed steps
required for silver staining procedures are undoubtedly
largely responsible for continued reliance on CBB staining
by many research laboratories. The maintenance of constant reaction temperatures is also critical for reproducibility, and seasonal fluctuations in ambient temperatures
may significantly impact results [23]. Due to the inherently
complex nature of silver staining procedures, spot intensities may vary by as much as 20% from run to run [49]. In
addition, the linear dynamic range of the stain is quite
poor [50]. Standard silver staining protocols almost invariably use glutaraldehyde and formaldehyde, which alkylate
a- and e-amino groups of proteins. This seriously hampers the analysis of proteins from gels by Edman-based
protein sequencing, though analysis by mass spectrometry can be implemented successfully if glutaraldehyde is
omitted from the staining procedure [51]. Such modified
silver stain-minus protocols are characterized by
decreased staining sensitivity and uniformity as well as increased gel background. Using the modified silver staining procedures, some have reported that the advantage

of higher detection sensitivity compared with CBB staining is counteracted by inferior sequence coverage in peptide mass fingerprinting experiments [52]. Sequence coverage refers to the percentage of protein accounted for in
the final peptide mass profile. Using a modified silver
staining procedure, abundant proteins can be expected to
yield roughly 1134% sequence coverage while CBB will
typically achieve values of 3067% [52]. A series of peaks
ranging from 1229 to 1670 Da, distributed in 44 Da increments, have also been observed using the modified silver
stain procedure and attributed to polymers generated in
the sample [52]. Additionally, silver-stained spots usually
show reduced peak intensity profiles compared with CBB
or zinc-imidazole reverse-stained material [52]. Low
sequence coverage of silver-stained proteins is thought to
arise from their Schiffs-base modification of free amino
groups by formaldehyde during the staining process. Unfortunately, formaldehyde can not be omitted from present silver stain protocols. The additional step of destaining
silver-stained gel bands with potassium ferricyanide and
sodium thiosulfate prior to enzymatic digestion has been
shown to reduce background interference and suppression of signals for MALDI-TOF peptide mass analysis
[41].
Among the common nonfluorescent detection methods,
zinc-imidazole reverse staining appears to have the fewest drawbacks with respect to use in protein mass profiling experiments [53, 54]. Sequence coverage is generally
equivalent to or better than that obtained after CBB staining [52]. Since gels are not fixed, peptide yields are
expected to be excellent using the reverse staining method, and indeed yields in the range of 9098% have been
documented using crushed polyacrylamide gel pieces
[29, 55]. While silver stain only provides a linear response
to protein amount in a restricted region of the low ng
range, the linear dynamic range of zinc-imidazole staining
is confined to a portion of the mg range (Fig. 1B; [50]).
Thus, the stain is completely inappropriate for quantitative
studies of protein expression. In addition, the actual stain
development step in reverse staining is quite short, typically 30 s, and prolonged staining obscures the clear protein zones.
2 Covalent labeling of proteins with

fluorophores
Numerous procedures for fluorescent derivatization of
proteins in gels and on transfer membranes have been
devised over the past three decades, utilizing a range
of fluorophores including fluorescamine, fluorescein isothiocyanate, monobromobimane, 2-methoxy-2,4-diphenyl3(2H)furanone (MDPF), p-hydrazinoacridine, N-(7-di-methylamino-4-methyl coumarinyl) maleimide, dichloro-triazinyl-
1127
with the succinimidyl esters of unsymmetrical cyanine

dyes, propyl Cy3 and methyl Cy5 [79, 80]. BODIPY-FL
maleimide has also been shown to be suitable for derivatization of cysteine residues in proteins prior to 2-D electrophoresis [81]. An alternative approach to prederivatization
labels primary amine residues with MDPF or 5-(4,6-dichlorotriazin-2-yl)aminofluorescein after IEF, but prior to SDS
gel electrophoresis to preserve the charge properties of
the proteins in the 2-D gel profile [82, 83].
2.1 Monobromobimane
Figure 1. The linear dynamic range of commonly

employed protein gel stains. (A) Comparison between
acidic silver nitrate stain and SYPRO Ruby Protein Gel
Stain. 11000 ng of bovine carbonic anhydrase II was
separated by SDS-polyacrylamide gel electrophoresis
and stained with either SYPRO Ruby Protein Gel Stain or
a commercially available acidic silver nitrate stain. Staining intensities were quantified using a Molecular Imager
FX system (Bio-Rad Laboratories). The line shows quantitation data obtained with the fluorescent stain (r =
0.9870). The superimposed gray bar shows the region of
linearity obtained with the silver stain method (r =
0.9916). (B) Linear dynamic ranges of common nonfluorescent protein detection methods compared with a fluorescent protein stain. The response of each stain was
evaluated using bovine carbonic anhydrase II, though
similar results were obtained when evaluating a range of
other proteins present in broad range molecular weight
markers. The graph is plotted using a logarithmic scale to
facilitate simultaneous viewing of the low ng through mg
regions. The linear region of the fluorescent stain encompasses the linear regions of all the other stains combined.
amino-fluorescein, and dansyl chloride [33, 5683]. Only

few approaches to covalent derivatization of proteins with
fluorophores have successfully been applied to protein
samples prior to their separation by 2-D gel electrophoresis, however. Cysteine residues have been modified with
monobromobimane and lysine residues have been labeled
Reactive halomethyl reagents, such as monobromobimane and monochlorobimane, mainly derivatize sulfhydryl-containing cysteine residues by nucleophilic substitution, though at alkaline pH they may also cross-react
with amines and imidazole nitrogens of histidine residues
[84]. Monobromobimane is generally preferred over
monochlorobimane because it reacts with thiol groups at
a 40-fold higher rate [84]. The probe has been used
extensively in the analysis of protein thiols and disulfides
using SDS-polyacrylamide gel electrophoresis [77, 85
88]. The covalent protein adduct resulting from alkylation
of cysteine residues by monobromobimane fluoresces
with an excitation maximum of 385 nm and emission maximum of about 470 nm [76]. After 2-D gel electrophoresis,
proteins may be viewed as turquoise bands using a
302 nm or 365 nm UV transilluminator. Alternatively, a
xenon arc light source combined with appropriate excitation and emission filters may be used to image the fluorescent bands. For example, the Wallac Arthur 1442
Multi-Wavelength Fluorimager (EG&G Wallac, Cambridge, England) may be used to visualize monobromobimane-labeled proteins in gels.
Monobromobimane was first used for prelabeling protein
sulfhydryl groups prior to 2-D gel electrophoresis in studies designed to analyze spermatozoa motility and flagellar
straightness [89]. Labeling was performed with intact
spermatozoa prior to their solubilization in electrophoresis
lysis buffer. Since monobromobimane is membrane-permeable, the fluorescent probe labels all reduced sulfhydryl groups in the cells but not oxidized groups [90, 91].
By comparing gels of monobromobimane-labeled samples with CBB-stained gels, the authors noted that numerous proteins in spermatozoa are labeled by both methods
but also that many proteins do not contain sufficient
amounts of reduced sulfhydryl groups for visualization
with monobromobimane. The selectivity of cysteine labeling was established by demonstrating a significant
decrease in protein fluorescence when cells were pretreated with iodoacetamide and a substantial increase
when cells were pretreated with dithiothreitol prior to
labeling with monobromobimane.
1128
W. F. Patton
Subsequently, prelabeling with monobromobimane was

introduced as a generalized method for visualization of
total protein profiles in 2-D gel electrophoresis [76, 79].
In order to obtain maximum fluorescence labeling of
sulfhydryl-containing proteins, monobromobimane was
added directly to solubilized cellular proteins in lysis buffer
containing 9 M urea, 4% Nonidet P-40, 2% carrier ampholytes, and 10 mM dithiothreitol. Roughly a 10% increase
in the number of protein spots detected on 2-D gels of
monobromobimane-labeled cellular proteins was noted in
comparison to silver-stained gels. In a more recent publication, a direct comparison of silver-stained, monobromobimane-labeled and [35S]methionine-labeled 2-D gels is
shown [92]. It is readily apparent from the protein profiles
displayed that monobromobimane detects many proteins
not visible by the other labeling methods but also that
many proteins labeled by the other techniques are absent from the monobromobimane-labeled samples. The
[35S]methionine-labeled protein profile is quite similar to
the silver-stained protein profile.
2.2 MDPF
MDPF, a fluorophore related to fluorescamine, reacts with
protein primary amine groups to form covalent Schiffs
base linkages. The dye is maximally excited at 385 nm
and maximally emits at 480 nm [82]. Thus, the dye-protein complex may be detected by similar methods as used
for the detection of monobromobimane-labeled proteins.
Initially, MDPF was used to covalently label proteins in
order to allow their subsequent quantitation after SDSpolyacrylamide gel electrophoresis [61, 93]. The approach was found to be superior to CBB staining followed
by densitometric scanning of gels [93]. As little as 1 ng of
protein is detectable using MDPF and the linearity of signal extends to 500 ng of derivatized protein [61]. MDPF
has also been employed to prederivatize proteins (prior to
SDS-polyacrylamide gel electrophoresis) followed by electroblotting [93]. Using 23 min film exposures, roughly 5 ng
of prederivatized protein was detectable in the gels while
100200 ng of protein could be visualized on nitrocellulose
membranes after electroblotting. The cited detection sensitivity on nitrocellulose membranes is no better than one
would expect to obtain using Amido Black or CBB dye.
Since MDPF reacts with e-amino groups of lysine residues and a-amino groups of N-termini of proteins to block
their native positive charges, this reagent is completely
unsatisfactory for prederivatization of proteins prior to 2-D
gel electrophoresis. However, MDPF may be used to derivatize proteins directly in IEF gels, prior to their separation by SDS-polyacrylamide gel electrophoresis [82, 83].
Similarly, the fluorophore 5-(4,6-dichlorotriazin-2-yl)aminofluorescein may be used to derivatize proteins in IEF

gels [83]. Alternatively, the proteins may be derivatized
with either fluorophore after both dimensions of separation are completed [82]. Typically, as little as 60 ng of protein may be visualized with MDPF using a cooled CCD
camera and exposure times of about 50 s. A linear response ranging from 50 to 300 ng has been reported [82].
A method for postelectrophoretic derivatization and
detection of proteins electroblotted to PVDF membranes
has been devised that uses a nonenzymatic, chemiluminescent reaction comprised of bis (2,4,6-trichlorophenyl)
oxylate and hydrogen peroxide for visualization of electroblotted or dot-blotted proteins covalently labeled with
MDPF [94]. Direct fluorescence detection of MDPFlabeled proteins on PVDF membranes using a 302 nm
UV transilluminator was later determined to be simpler,
less time-consuming, and more sensitive than the original
chemiluminescent method [95]. The direct fluorescence
method is capable of detecting 510 ng of protein and is
compatible with chemiluminescent immunodetection.
However, proteins labeled with MPDF must be viewed
using wet PVDF membranes as the fluorescence signal
decreases 500-fold upon drying.
MDPF has also been reacted with dodecyl or octyl amine
to produce hydrophobic derivatives suitable for noncovalent detection of proteins in polyacrylamide gels [96]. After
electrophoresis, gels are incubated in 30% methanol for
30 min and then stained in 0.5% fluorophore in water for
1 h. Upon UV illumination, octyl MDPF displays fluorescent bands with a limit of detection of roughly 20 ng.
Dodecyl MDPF requires an additional overnight incubation in 5% methanol to reduce background staining before
a similar detection sensitivity is achieved. While staining
with these dyes is also possible by adding them to cathode buffer during electrophoresis, detection sensitivity is
reduced to 4080 ng per protein band. The detection sensitivities of the derivatized MDPF dyes are thus quite similar to CBB staining and to date they have not been
offered commercially.
2.3 Propyl Cy3/methyl Cy5

Succinimide ester-containing reagents are commonly
used to modify primary amines [97, 98]. These compounds react with nucleophiles, subsequently releasing
the N-hydroxysuccinimide (NHS) group to form an acylated product. In proteins, these reagents react almost
exclusively with a-amines at the N-termini of proteins and
with e-amines of lysine residues to form stable amide linkages that are fairly resistant to hydrolysis. Succinimydyl
esters of propyl Cy3 and methyl Cy5 dyes have been
utilized to prelabel two different protein samples prior to
running them on the same 2-D gel [80]. This permits two
samples to be run under identical electrophoretic condi-

tions in a type of differential display format. The two dyes
have been carefully designed so that they migrate to the
same position on a 2-D gel. This entails regenerating the
positive charge of the lysine residue upon reaction with
proteins. Though an ionizable primary amino group from
lysine is replaced with a nonionizable quartenary amino
group from the dye, with a predicted pKa shift from 9 to
14, this is not a significant alteration within the pH range
of 310 that is normally used for IEF separations. At the
alkaline extreme of an immobilized pH gradient gel, however, some differences in protein migration are expected
to occur. The molecular weight of the dye molecules are
closely matched as well, though the underivatized proteins in the sample migrate with a lower apparent molecular weight. While Cy5 has a longer linker region between
the two indole groups than Cy3, a methyl moiety on Cy5
is replaced with a propyl moiety on Cy3 to compensate
for the resulting molecular weight difference. The fluorescence characteristics of the two dyes are not as closely
matched. While Cy3 dye has a quantum yield of 0.15 and
molar extinction coefficient of 150 000 M1 cm1, Cy5 dye
has a quantum yield of 0.28 and molar extinction coefficient of 250 000 M1 cm1. Since Cy3 is considerably
dimmer than Cy5, modest differences in spot abundance
between two samples must be determined ratiometrically
using other spots in each pattern as internal calibration
standards. To date, only evaluations of qualitative (plus/
minus) differences between protein profiles have been
demonstrated using the technology [80].
Cy3-derivatized proteins maximally excite at about
550 nm and maximally emit at 570 nm. Their counterparts, the Cy5-derivatized proteins, maximally excite at
650 nm and maximally emit at 670 nm. Consequently, the
use of these dyes is restricted to a handful of specialized
instruments such as the Wallac Arthur 1442 Multi-Wavelength Fluorimager and Fuji FLA 3000 G Fluorescent
Image Analyzer. The dye pair can not be visualized effectively by simply placing a gel on a UV transilluminator.
This presents problems for direct excision of protein spots
from the gels destined for mass spectrometry as the primary commercial instruments for detecting the dyes do
not allow the user to directly view the gel. A homemade
viewing system may be constructed using 100 W halogen
lamps and appropriate filters, but the protein/cyanine dye
conjugates are so dim that a 30 min camera exposure
time is required to view 1 ng of protein [80]. Thus, only the
most abundant components of a sample may be viewed
directly by eye. Excision of spots from Cy3/Cy5 labeled
protein profiles is probably best accomplished indirectly
as is often done in autoradiography. The gel should be
placed on a 1:1 size ratio printout of the fluorescent image
produced from an appropriate gel documentation system
and spots cut out with reference to the mask.
1129
2.4 Merits of derivatizing amino versus

sulfhydryl groups
There is no completely satisfactory method for determining absolute protein amount by image analysis of 2-D gels
without a priori knowledge of that proteins identity and
sequence. Biases are introduced due to inequalities in the
number of covalently labeled residues present in a given
protein. In equilibrium, metabolic radiolabeling experiments with [35S]methionine, the number of methionine
residues in a given protein, the radioactivity of the protein
and the specific activity of the radiolabel normalized per
methionine residue must be known in order to accurately
determine a proteins actual abundance [99]. Similarly,
the labeling of a known number of cysteine residues to
generate conjugates, which each have an identical quantum yield, is a prerequisite for rigorous quantitation of proteins after fluorescent labeling with monobromobimane
[100].
An ideal detection technology might be expected to interact with each amino acid in a polypeptide chain equally
well, so that a protein molecule that is twice as large as
another would generate twice as much signal. Probably
the closest anyone has come to achieving such an idealized detection technology is through radiolabeling with a
mixture of 16 different 14C-L-amino acids [101]. Given that
the targeting of one particular amino acid residue for
covalent labeling is not a completely satisfactory method
for protein quantitation, the next issue that should be
addressed is whether some amino acid residues are
more suitable than others for the global analysis of protein
expression. Cysteine residues appear to be especially
poor targets for uniform labeling of proteins when compared with methionine or lysine residues. Some proteins
contain an especially high abundance of cysteine residues such as serum albumin (35 residues), low-density
lipoprotein receptor (61 residues), ultra-high sulfur keratin-associated protein I (93 residues), testicular metalloproteinase-like, disintegrin-like, cysteine-rich protein
(tMDC I; 42 residues), zyxin (21 residues), and vascular
endothelial cell growth factor (VEGF; 16 residues). Others
are completely devoid of cysteine residues such as myoglobin, concanavalin A, cytochrome b5, thermolysin, lentil
lectin, staphylococcal nuclease, substilin BPN, and
bovine carbonic anhydrase II.
The shortcomings of labeling cysteine residues for global
protein quantitation become readily apparent upon evaluation of protein standards commonly used in gel electrophoresis (Fig. 2, Table 1). Though hardly representative
of an entire proteome, I have found this proteomic microcosm surprisingly effective at revealing fundamental
flaws in protein detection strategies. A number of protein
1130
W. F. Patton
Figure 2. Protein detection by

covalent labeling of cysteine
residues with monobromobimane. Off-the-shelf, broad, pI
calibration standards (Amersham Pharmacia Biotech) were
heat-denatured in 200 mM Tris,
pH 7.8, 1% SDS, 3 mM EDTA,
3 mM dithiothreitol and then
cooled to room temperature,
labeled with 6 mM monobromobimane, diluted in water and
separated on a Multiphor II unit
using Ampholine PAGplate gels
(pH 3.59.5). The standards used
were: trypsinogen (pI 9.30), basic
band of lentil lectin (pI 8.65), middle band of lentil lectin (pI 8.45),
basic band of horse myoglobin (pI
8.45), acidic band of horse myoglobin (pI 6.85), human carbonic anhydrase B (pI 6.55), bovine carbonic anhydrase B (pI 5.85),
b-lactoglobulin A (pI 5.20), soybean trypsin inhibitor (pI 4.55), and amyloglucosidase (pI 3.50). (A) Proteins stained with SYPRO
Ruby IEF Protein Gel Stain. (B) Proteins prelabeled with monobromobimane. While SYPRO Ruby IEF Protein Gel Stain readily
reveals all of the marker proteins, numerous proteins are not visualized with monobromobimane. Arrow indicates a stained carrier ampholyte zone. Figure courtesy of Kiera Berggren and Tom Steinberg, Molecular Probes.
Table 1. Correlation between labeling targets and protein molecular weight

Protein
Rabbit skeletal muscle myosin

Human a2-macroglobulin
Escherichia coli bgalactosidase
Rabbit muscle phosphorylase b
Bovine serum albumin
Porcine fumarase
Chicken egg white ovalbumin
Porcine pepsin
Bovine carbonic anhydrase
Rabbit trisephosphate
isomerase
Soybean trypsin inhibitor
Bovine b-lactoglobulin
Horse myoglobin
Chicken egg white lysozyme
Bovine aprotinin
Insulin beta chain
Apparent Methionine
molecular residues
mass
(kDa)
Cysteine
residues
Lysine
residues
Lysine +
arginine +
histidine
residues
200
180
116
54
25
24
16
24
16
190
89
20
332
174
120
97
66
48.5
45
34.7
31
27
21
5
17
17
4
3
2
9
35
3
6
6
0
5
48
60
30
20
1
18
21
133
103
59
42
4
38
33
21.5
18.4
17
14.4
6.5
3.5
2
5
2
2
2
0
4
7
0
8
4
2
10
16
19
6
4
1
21
21
32
18
8
4
Linear correlation coefficient
R2 = 0.8283
R2 = 0.3830
R2 = 0.7524
R2 = 0.8942
Representative detection
method
[35S]methionine
labeling
MonobromoSilver staining,
Colloidal CBB,
bimane labeling
MDPF, Cy3/Cy5 SYPRO Ruby
labeling
dye staining

detection technologies that have been published in the literature can immediately be dismissed by simply evaluating a gel containing broad-range molecular weight or isoelectric focusing markers (Fig. 2). As demonstrated in
Table 1, the number of cysteine residues does not correlate well with the molecular weight of protein standards. In
fact, two of the common protein standards are devoid of
cysteine groups and one contains an excessive number
of these residues. Monobromobimane is thus more suitable for evaluating the redox state of proteins in cells than
as a general protein label [89]. Methionine and lysine residues are vastly superior to cysteine for general protein
detection, though noncovalent dyes that bind to all basic
amino acid residues such as colloidal CBB dye and
SYPRO Ruby dye provide an even better correlation with
molecular weight.
2.5 Covalent derivatization: caught between a

rock and a hard place
Intuitively, many scientists shy away from the concept of
prelabeling proteins with fluorophores prior to gel electrophoresis. After all, according to one theory, unnecessary
modification of amino acid residues in proteins ought to
be avoided. Indeed, monobromobimane, MDPF, and
Cy3/Cy5, the covalent derivatization technologies that
have been advocated for use in 2-D gel electrophoresis,
share certain limitations that should be kept in mind when
performing particular types of proteomic investigations.
Despite attempts to minimize differences in the electrophoretic behavior of underivatized and derivatized proteins on 2-D gels, anomalous migration of proteins invariably does occur. Retarded mobility of proteins has been
observed with all of the cited labeling techniques, though
most particularly with the lysine-modifying agents [80, 82,
83]. These changes in protein mobility are most readily
observed among the lower molecular weight proteins due
to the logarithmic nature of electrophoretic separations.
Often a derivatized spot is observed to possess a streaky
comet tail that extends to higher molecular weight [82].
This may very well be ascribable to the altered SDS binding characteristics of the labeled proteins.
Based upon data obtained from cryo-electron microscopy
and X-ray scattering, SDS appears to bind along an
unfolded polypeptide chain as a series of 6.2 nm spherical
micelles with center-to-center distances of 712 nm [102,
103]. For comparison, pores in polyacrylamide gels are
estimated to range from 0.5500 nm, depending upon
matrix composition [104107]. The necklace-like structure
of the SDS/protein complex is characterized by the polypeptide backbone and polar side chains interacting with
the sulfate head groups on the surface of the micelle,
while the hydrocarbon chain of the SDS molecules and
the hydrophobic side chains of the protein are localized to
1131
the interior of the micelle [102]. The nucleation sites for

the micelles have not yet been determined, though basic
proteins such as histone H5 are notable in having larger
micelle complexes with amino acid side chains projecting
outwards from the micelle rather than into the core [102].
The fluorophore, 1-anilino-8-napthalene sulfonate (ANS),
is often used as a hydrophobic probe for examining the
nonpolar characteristics of proteins. Recently, it has conclusively been demonstrated that the primary binding
mechanism of this fluorophore is through ion pair formation with the cationic amino acid residues lysine, arginine,
and histidine [108]. Similar behavior has been observed
with other organic sulfonate and sulfate ligands, including
detergent sulfates such as SDS [108110]. In my own
laboratory we have noted that SYPRO Orange dye, which
binds to proteins via interaction with SDS micelles, primarily labels amino acid homopolymers of lysine, arginine, and histidine. Omission of SDS completely prevents
labeling of the basic amino acid polymers. In a similar
vein, histones contain an unusually large number of lysine
and arginine residues. The fluorescent staining of histones with the hydrophobic dye Nile Red may be achieved
in solution using much lower concentrations of SDS than
with other proteins due to stronger binding of the detergent [111]. Together, these observations suggest that
basic amino acids play a fundamental role in binding
anionic detergent and are therefore crucial to SDS-mediated electrophoretic separations. Modification of lysine
residues in particular is likely to cause changes in protein
migration arising from altered SDS binding as well as due
to the modest increase in weight accompanying attachment of the fluorophore.
Besides altering the SDS-binding properties of basic
amino acid residues, the covalent attachment of a fluorophore dramatically alters the hydrophilic nature of particular amino acid residues like lysine. Natively hydrophobic
proteins are conspicuous in their absence from current 2-D
gel profiles and it is likely that other proteins will be lost
from the patterns after their derivatization with hydrophobic dyes [112]. Even the simple substitution of a single glutamine residue with a leucine residue in the 20 000 Da protein a-crystallin is known to alter the mobility of the protein
by 3% in SDS-polyacrylamide gel electrophoresis [113].
The unsulfonated Cy3/Cy5 dyes in particular dramatically
increase a proteins overall hydrophobicity. Consequently,
protein solubility is substantially reduced after labeling.
This is somewhat of a catch-22 situation: in order to obtain
the maximum detection sensitivity one should completely
derivatize a protein at all lysine residues with Cy3/Cy5, but
to minimize solubility problems one should label as few
residues as possible. Operationally, Unlu and co-workers
[80] found that a satisfactory compromise is to label a van-
1132
W. F. Patton
ishingly small number of proteins in a sample (on average

12% of the proteins) and detect them with a sensitive
cooled CCD camera. Despite the very low degree of substitution, the modified proteins migrate to a different position than the unmodified proteins in the gel. Due to the differences in mobility, one must excise regions of a gel that
are biased by about one spot diameter to lower molecular
weight in order to obtain material for analysis by mass
spectrometry or Edman sequencing, in essence guessing
where the unmodified protein migrates and substantially
increasing the possibility that the wrong protein will be
characterized. Also of note, the effective resolution of a 2D gel is reduced by half due to the duplication of protein
spots in the molecular weight dimension. On the positive
side, with this approach, the total number of amino acid
residues deri-vatized in the sample is quite small and thus
of little consequence with respect to amino acid analysis,
Edman-based sequencing, or peptide-mapping studies
[60, 80, 95].
One solution to the hydrophobicity problems associated
with the cyanine dyes might be to synthesize more soluble versions of the fluorophores [80]. While derivatization
of 100% of the lysine residues in a protein with these
new, hypothetical cyanine dyes could improve detection
sensitivity, cyanine dyes are quite large and the molecular
weight of a typical protein would increase by roughly
4 00010 000 Da depending upon whether the protein
represented an a, b, a + b or a/b protein structural class.
Also, based upon studies with MDPF, it is apparent that if
proteins are destined for N-terminal Edman sequencing,
labeling must be performed with a suboptimal dye concentration for a short duration so that the primary amines
are not all consumed in the reaction. For MDPF these
modified labeling conditions result in fluorescent detection
that is no more sensitive than standard CBB staining procedures [95]. Higher degrees of dye substitution also
require adjustments in the interpretation of mass spectrometry data. For instance, identification of proteins by
mass spectrometry after monobromobimane labeling is
complicated by molecular mass increases of 190 Da for
all derivatized cysteine residues [50, 92]. If the derivatization reaction is not 100% efficient, the unmodified peptide
is also present in the spectrum. In addition, side reactions
with other functional groups such as lysine and histidine
residues as well as altered specificity of trypsin arising
from its interaction with unnatural amino acid residues
can make interpretation of spectra nearly impossible [50].
3 Noncovalent fluorescent staining of

proteins
A number of fluorophores such as ANS, bis (8-toluidino1-naphthalenesulfonate) (bis-ANS), 9-diethylamino-5Hbenzo[a]phenoxazine-5-one (Nile Red), SYPRO Orange

dye SYPRO Red dye, and SYPRO Tangerine dye interact
with proteins noncovalently [111, 114126]. These fluorophores are virtually nonfluorescent in aqueous solution
but become fluorescent in nonpolar solvents or upon
association with SDS-protein complexes. SDS binds to
protein with a fairly constant stoichiometry, suggesting
that protein quantitation based upon an interaction with
the detergent should be more reliable than one based
upon interactions with primary amines alone. As a whole,
protein detection sensitivity with many of the older fluorophores in this category has been fairly poor and few are
routinely used in research laboratories today. Within this
family of dyes only the SYPRO dyes are exceptional in
that they deliver detection sensitivities that are competitive with colloidal CBB staining and rapid silver staining
methods.
In addition to the dyes that intercalate into SDS micelles,
a newly developed family of luminescent transition metal
complexes, SYPRO Ruby and SYPRO Rose dyes, allow
sensitive fluorescence detection of proteins after IEF gel
electrophoresis, SDS-gel electrophoresis, 2-D gel electrophoresis, and electroblotting [43, 50, 127129]. The fluorophores bind to proteins by a mechanism that is quite
similar to CBB staining and are as sensitive as the best
silver staining procedures available. The dyes are superior to silver staining in terms of linear dynamic response
and compatibility with downstream microchemical protein
characterization techniques. Some important characteristics of noncovalent dyes that interact directly with proteins
or indirectly via the SDS micelle are summarized in Table
2.
3.1 Nile Red dye

Nile Red dye was originally used as a selective fluorescent vital stain for visualizing intracellular lipid droplets by
flow cytometry and fluorescence microscopy as well as
for quantifying lipids on thin-layer chromatograms [130].
More recently, the dye has been adapted to detect bacterial polyhydroxyalkanoic acids in Gram-negative bacteria
[131, 132]. Nile Red dye is maximally excited at either
300 or 540 nm and emits at approximately 640 nm [111].
This fluorescent oxazone dye appears to be solubilized
by SDS-protein complexes in a manner similar to other
oil-soluble dyes [133]. The detection sensitivity of 5
50 ng for Nile Red staining of proteins in SDS-polyacrylamide gels is slightly better than detection obtained with
CBB staining, and the dye is compatible with standard
Edman sequencing and immunodetection procedures
[95, 120, 121, 134]. Nile Red dye may be used to prestain
serum lipoproteins in native agarose gel electrophoresis,
but some staining of other hydrophobic proteins also
occurs [135138]. For this reason, the dye is best used to

evaluate samples that have been pretreated to remove
serum albumin. Detection sensitivity for low density lipoproteins is in the vicinity of 2.5 mg.
Nile Red dye is nearly insoluble in water and stock solutions of the dye are prepared in dimethyl sulfoxide [111].
Dye staining of proteins in SDS-polyacrylamide gels is
typically performed in a solution with a final composition
of 2% dimethyl sulfoxide in water [121]. Thus, unlike CBB
and silver staining solutions, which contain fixatives such
as methanol and acetic acid that preclude effective transfer of stained proteins, the Nile Red staining solution
allows for ensuing protein transfer to PVDF membranes
by electroblotting [95, 121]. Esthetically, Nile Red dye
staining is a fairly unsatisfactory method for protein visualization. The insoluble nature of Nile Red often leads to
nonspecific precipitation of the dye on the surface of gels
and in the staining tray. Serial staining experiments of
proteins in gels with Nile Red, followed by MDPF labeling
on PVDF membranes demonstrate significant background fluorescence from absorbed Nile Red dye that
strongly interferes with detection of the MDPF label [95].
By using Wratten #3 and #47 cellophane filters (Kodak,
Rochester, NY), the Nile Red dye fluorescence can be
effectively screened out, allowing visualization of the
MDPF signal. Another problem associated with staining
proteins using Nile Red is that significant photobleaching
of the dye is observed even after brief exposure to UV illumination [111].
3.2 SYPRO Orange, Red and Tangerine dyes

SYPRO Orange, SYPRO Red, and SYPRO Tangerine
dyes (Molecular Probes) can detect proteins in SDS-polyacrylamide gels using a simple, one-step staining procedure that requires 3060 min to complete and does not
involve a destaining step [122126]. As with Nile Red
dye, binding is achieved through intercalation with SDS/
protein complexes. Though hydrophobic in character,
these SYPRO dyes are more water-soluble than Nile Red
and thus do not suffer from problems of nonspecific precipitation on gel surfaces and staining trays, though some
cosmetic speckling of fluorescent stain may be observed.
In contrast to many silver staining and reverse staining
methods, the SYPRO dyes do not stain nucleic acids or
bacterial lipopolysaccharides to a significant extent [123,
126]. Gels may be rapidly and completely destained by
incubation in an aqueous solution containing 30% methanol [122].
As little as 410 ng of protein can routinely be detected
with this group of SYPRO dyes, rivaling the sensitivity of
rapid silver staining techniques and surpassing the best
colloidal CBB staining methods available [122, 123, 126].
1133
Since all three dyes excite at roughly 300 nm, documentation of stained gels can readily be achieved by photography on a standard laboratory 302 nm UV transiluminator.
Alternatively, the dyes may be quantified with commercially available CCD camera-based image analysis workstations or a variety of laser scanners, providing a linear
dynamic range of three orders of magnitude. In addition
to the UV excitation peak, SYPRO Orange, Red, and
Tangerine dyes may be excited by visible light at approximately 470, 550, and 490 nm, respectively. The three
dyes maximally emit at 570, 630, and 640 nm, respectively. Thus, SYPRO Orange and Tangerine dyes are well
suited for imaging systems that incorporate 473 nm second harmonic generation (SHG) or 488 nm argon ion (Ar)
lasers as well as those that use 470 nm blue light emitting
diodes (LEDs) or 460 nm blue fluorescent light sources.
SYPRO Red dye is most suitable for 532 nm frequencydoubled neodymium-yttrium-aluminum-garnet (Nd-YAG)
laser sources as well as 543 nm helium-neon (He-Ne)
laser sources.
Coelectrophoretic staining may be accomplished by
including one of the SYPRO dyes in the running buffer of
SDS-polyacrylamide gels, but detection sensitivity is 4- to
8-fold poorer than postelectrophoretic staining [122]. After
electrophoresis, gels are briefly destained prior to visualizing proteins. SYPRO Orange, Red, and Tangerine dyes
may be employed to detect proteins in SDS-capillary gel
electrophoresis as well as in conventional slab gel electrophoresis [139]. Protein samples solubilized in 0.5%
SDS are noncovalently labeled with the SYPRO dyes
prior to electrokinetic injection at the cathodic end of
uncoated 50 mm ID capillaries filled with 8% linear polyacrylamide. Separation of proteins in the mass range of
2978 kDa is achieved in 18 min with as little as 73 pM
bovine serum albumin being detectable.
SYPRO Tangerine dye was developed to address specific shortcomings encountered in the use of the originally
introduced SYPRO Orange and SYPRO Red dyes [126].
Both SYPRO Orange and SYPRO Red dyes require 7%
acetic acid in the staining solution, which is problematic
when electroblotting, electroeluting, or measuring enzyme
activity is indicated. If acetic acid is omitted from the staining solution, proteins may be recovered from gels, but the
detection sensitivity obtained with SYPRO Orange and
SYPRO Red stains is substantially lower and significant
protein-to-protein variability in staining is observed [123].
SYPRO Tangerine stain is an environmentally benign
alternative to conventional protein stains that does not
require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained
in a wide range of buffers, including phosphate-buffered
saline or simply 150 mM NaCl. Since proteins may be
1134
W. F. Patton
stained using SYPRO Tangerine dye without the need for

harsh fixatives, they are easily eluted from gels or utilized
in zymographic assays, provided that SDS present during
electrophoresis does not inactivate them. This has been
demonstrated with in-gel detection of esterase activity
using a-naphthyl acetate and Fast Blue BB dye as well as
b-glucuronidase activity using ELF-97 b-D-glucuronide
[126]. The dye is also suitable for staining proteins in gels
prior to their transfer to membranes by electroblotting.
SYPRO Tangerine dye staining allows visualization of
proteins and excision of small regions of a gel or even of
individual bands, followed by their transfer to membranes
by electroblotting. This permits use of much smaller
amounts of transfer membrane and corresponding savings in immunodetection reagents [28]. As with reversestaining procedures, the gentle staining conditions used
with SYPRO Tangerine dye are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate and aspartate
residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry. With a
7% acetic acid staining solution, SYPRO Tangerine-dyestained gels display slightly higher background staining
than SYPRO Orange and Red dyes. Thus, the later dyes
are preferred choices for routine staining in a fixative solution.
SYPRO Orange, Red, and Tangerine dyes are not recommended for detection of proteins electroblotted to membranes or separated by IEF or nondenaturing gel electrophoresis. Fluorescent detection of proteins in IEF gels
has been accomplished using Nile Red dye and my group
finds that SYPRO Red, Orange, and Tangerine dyes can
also stain IEF gels in a similar manner ([121]; unpublished
observations). Such gels must be incubated in SDS prior
to staining since all four of these lipophilic dyes bind to
proteins indirectly through the anionic detergent. Analogous protocols may be used for detecting proteins in nondenaturing gels [123]. The disadvantages of using three
fluorescent dyes to stain IEF and nondenaturing gels are
that detection sensitivity is often poorer than standard
CBB staining and the incubation step in SDS is likely to
lead to some loss of protein. Similarly, the detection sensitivity of SYPRO Orange, Red, and Tangerine dyes in
2-D gels is somewhat poorer than simple, 1-D SDS-polyacrylamide gels. This is probably due to the mixed
micelles encountered in 2-D gel electrophoresis that contain nonionic detergents such as Triton X-100 as well as
carrier ampholytes. In the context of 2-D gels, high quality
silver staining procedures usually surpass these fluorescent dyes in terms of detection sensitivity, while colloidal
CBB offers a comparable level of sensitivity. However,
the fluorescent dyes are fully compatible with Edman-

based sequencing and mass spectrometry [126, 140,
141].
3.3 SYPRO Rose and SYPRO Ruby dyes

Certain d-block and lanthanide transition metal complexes such as those composed of ruthenium, rhenium,
osmium, platinum, europium, or terbium are intensely
luminescent. The transition metal chelate dyes, SYPRO
Ruby and SYPRO Rose stains, are complexes of ruthenium and europium, respectively, that bind avidly to proteins by a CBB-type mechanism primarily involving lysine,
arginine, and histidine residues. These dyes represent a
relatively new family of luminescent protein visualization
reagents developed to be fully compatible with modern
proteomics research [43, 50, 127129, 142]. Colorimetric
predecessors to the luminescent stains include Ferrozine/
iron, Ferene S/iron, bathophenanthroline disulfonate/iron,
pyrogallol red/molybdenum, and phthalocyaninetetrasulfonate/copper stains [127, 128, 142147]. The luminescent stains have been designed specifically for compatibility with commonly used microchemical characterization
procedures and do not contain extraneous chemicals
such as glutaraldehyde, formaldehyde, or Tween-20 that
are known to compromise such procedures. Luminescent
transition metal chelate stains are easily incorporated into
integrated proteomics platforms [3]. The most sensitive
luminescent stain, SYPRO Ruby stain, is more sensitive
and exhibits a broader linear dynamic range than the best
alkaline silver diamine and acidic silver nitrate staining
procedures available.
Bathophenanthroline disulfonate/europium complex (SYPRO Rose Protein Blot stain; Molecular Probes) is a
medium sensitivity stain that routinely detects 24 ng/
mm2 of applied protein as determined by slot-blotting,
which translates to about 1530 ng of gel applied protein
in routine electroblotting applications [127, 128]. The stain
can be visualized using 302 nm UV epi-illumination and
emits at 590 and 615 nm. The stain is readily removed
from proteins by incubating at neutral to mildly alkaline
pH. Compatibility with immunoblotting, lectin blotting,
Edman sequencing, and mass spectrometry make SYPRO Rose stain a good, routine protein detection reagent
that is nearly as simple to use as Ponceau S stain [127].
Limitations of the dye are that it can only be visualized
using UV epi-illumination and, in addition to the desired
red fluorescence at 595 and 615 nm, the stain exhibits
intense blue fluorescence at 450 nm due to uncomplexed
ligand.
SYPRO Rose Plus Protein Blot stain was subsequently
developed to overcome some of the limitations encountered with the original electroblot stain. SYPRO Rose Plus

Protein Blot Stain is an improved europium-based metal
chelate stain that is roughly ten times brighter than the
bathophenanthroline disulfonate/europium stain. The
intense blue fluorescence from uncomplexed ligand observed in the original stain has been eliminated by
employing a thermodynamically more stable europium
complex and the stain can be readily visualized by UV
epi-illumination. Since the original stain displays two
emission peaks of roughly the same intensity at 595 and
615 nm, a 24 nm emission window must be used to collect the signal. By comparison, SYPRO Rose Plus Protein
Blot Stain displays a single emission peak at 615 nm with
razor-sharp 8 nm half band width. Just as with the bathophenanthroline disulfonate/europium stain, SYPRO Rose
Plus stain is easily removed by increasing solution pH.
The stain is fully compatible with MALDI-TOF mass spectrometry, biotin/streptavidin detection systems and immunoblotting. Just like its predecessor, however, the new
dye can not be utilized with laser-based gel scanners
since it is not excited by visible light. Our group has successfully imaged dye-stained proteins using the xenon
arc lamp-based Wallac Arthur 1442 Multi-Wavelength
Fluorimager equipped with a 390 +/ 35 nm excitation
filter, a configuration commonly used for excitation of
monobromobimane.
SYPRO Ruby dye is a proprietary ruthenium-based metal
chelate stain developed to address the limitations of the
SYPRO Rose dyes. SYPRO Ruby Protein Blot stain (Molecular Probes) permanently stains electroblotted proteins
on nitrocellulose and PVDF membranes with a detection
sensitivity of 0.251 ng of protein/mm2 in slot-blotting
applications. Approximately 28 ng of protein can routinely be detected by electroblotting, which side-by-side
comparisons have demonstrated to be as sensitive as
colloidal gold stain [43]. While colloidal gold staining
requires 24 h, SYPRO Ruby dye staining is complete in
15 min. The linear dynamic range of SYPRO Ruby Protein Blot stain is vastly superior to colloidal gold stain,
extending over a 1000-fold range. The dye can be excited
using a standard 302 nm UV transilluminator or using
imaging systems equipped with 450, 473, 488 or even
532 nm lasers. The dye emits maximally at about 618 nm.
Unlike colloidal gold stain, SYPRO Ruby stain does
not interfere with mass spectrometry, Edman-based
sequencing, or immunodetection procedures [43].
SYPRO Ruby Protein Gel stain and SYPRO Ruby IEF
Protein Gel stain allow one-step, low-background staining
of proteins in gels without resorting to lengthy destaining
steps. The linear dynamic range of these dyes extends
over three orders of magnitude, thus surpassing silver
and Coomassie blue stains in performance. An evaluation
of 11 protein standards ranging in isoelectric point from
1135
3.5 to 9.3 indicates that SYPRO Ruby IEF gel stain is 3

30 times more sensitive than highly sensitive silver stains
[129]. Proteins that stain poorly with silver stain techniques are often readily detected by SYPRO Ruby dye.
The fluorescent gel stains can be visualized using a wide
range of excitation sources commonly used in image
analysis systems including a 302 nm UV transilluminator,
473 nm SHG laser, 488 nm argon-ion laser, 532 nm NdYAG laser, xenon arc lamp, blue fluorescent light bulb or
blue LED. Both dyes maximally emit at about 610 nm.
Though more sensitive than SYPRO Orange, Red, and
Tangerine dyes, optimal staining is somewhat slower,
requiring about 4 h to complete. Similar to colloidal Coomassie blue stain but unlike silver stain, SYPRO Ruby
dye stains are end-point stains. Thus, staining times are
not critical and staining can be performed overnight without gels overdeveloping. SYPRO Ruby Protein Gel stain
and SYPRO Ruby IEF Protein Gel stain are ideally suited
for use in the identification of proteins by peptide mass
profiling using MALDI-TOF mass spectrometry [43, 50,
126].
3.4 Whats old is new again

From the perspective of this reviewer at least, it is interesting to note that we have come full circle with respect to
the detection of proteins in gels and on blots. CBB staining has withstood the test of time and remains the most
popular protein staining technology ever devised for gel
electrophoresis. During its nearly 40-year history in the
research laboratory, the only substantive complaints
about the method, other than a propensity for producing
blue fingers, have centered upon sensitivity and linear
dynamic range. Even in this new era of proteomics, CBB
is the benchmark stain used to assess the performance of
all other detection technologies, and alternatives such as
silver staining often do not quite measure up [52]. Published reports of new colorimetric stains have appeared
sporadically in the literature, some of them being covalent
derivatization techniques using reactive dyes such as
Remazol Brilliant Blue, Drimarene Brilliant Blue, dabsyl
chloride, or Uniblue A [148154]. Such approaches have
not gained widespread acceptance, except in restricted
applications such as the generation of prestained protein
molecular weight standards [155]. The mobility defects
associated with covalently derivatizing proteins are well
recognized and such commercialized standards are usually supplied with a list of revised molecular weight values
[152].
In the realm of fluorescence detection technologies, lessons learned by the previous generation of electrophoresis scientists are being rediscovered by a new generation
of practitioners. A number of strategies for fluorescence
1136
W. F. Patton
detection of proteins is possible, but a stain that can be

considered conceptually as a fluorescent CBB stain offers
the broadest applicability. SYPRO Ruby dyes are compatible with a wide range of light sources, allowing fluorescence detection by eye or using almost any available
imaging platform, from instant camera to laser scanner.
Since the dyes bind to proteins noncovalently, no protein
modifications are produced, greatly simplifying interpretation of peptide mass profiles generated by mass spectrometry [43, 50, 126]. In addition, the dyes are appropriate for staining proteins in a wide range of procedures
including IEF gel electrophoresis, SDS-gel electrophoresis, 2-D gel electrophoresis, and electroblotting [43, 50,
126]. Since the SYPRO Ruby dyes are not present during
electrophoresis, aberrant migration of proteins is avoided.
Spots can be concisely excised from gels, minimizing
cross-contamination of proteins from other regions of the
gel.
much as 1000-fold and extends the time of light emission

to about 60 min [158, 159]. Typically, Western blot systems employing this approach, such as the ECL reagent
(Amersham Pharmacia Biotech, Uppsala, Sweden) are
capable of detecting as little as 110 pg of target protein.
Alternative azine and acridan enhancer systems have
been developed more recently that extend the duration of
light emission to as long as 48 h and allow fg levels of protein detection. These systems include SuperSignal
(Pierce Chemical, Rockford, IL), ECL Plus (Amersham
Pharmacia Biotech), and Lumi-Light Plus (Roche Molecular Biochemicals, Indianapolis, IN) Western blot kits [160].
Similarly, chemiluminescence technology has been
devised for the detection of alkaline phosphatase-conjugated antibodies. For example, CSPD substrate (Tropix,
Bedford, MA) is a 5-chloro-derivative of adamantyl aryl
dioxetane phosphate 1,2-dioxetane that is used in this
capacity [161163].
Finally, because SYPRO Ruby and SYPRO Rose dyes

incorporate luminescent transition metals as fluorophores, they could potentially be used for time-resolved
imaging of gels and blots in the not too distant future. This
is expected to increase detection sensitivity by as much
as two to three orders of magnitude relative to the prompt
detection of the stains currently performed [156]. If such
improvements in performance are realized, these fluorescent stains should be able to detect low pg instead of low
ng amounts of protein. Instrumentation capable of timeresolved imaging, such as the LEADseeker (Amersham
Pharmacia Biotech, Amersham, UK) are only beginning
to appear in the market place, and their continued refinement and propagation are all but guaranteed as imagebased, high-throughput screening assays are implemented in the pharmaceutical industry.
4.2 Direct fluorescence detection
4 Luminescence immunodetection on
electroblots
4.1 Dominance of chemiluminescence
detection
Luminescent detection of specific proteins after immunoblotting is rarely performed by direct fluorescence procedures. Instead, a chemiluminescence approach is usually
adopted. Horseradish peroxidase-conjugated antibodies
may be detected through oxidation of diacylhydrazides,
such as luminol, in the presence of hydrogen peroxide
and a phenolic enhancer under alkaline conditions [157].
The resulting excited-state product quickly decays to
ground state and emits blue light. Phenolic enhancers
such as para-iodophenol act as radical transmitters between the horseradish peroxidase-generated free radical
and the luminol, which increases light emission by as
Until recently, direct fluorescence immunodetection has

suffered from very poor detection sensitivity compared
with enhanced chemiluminescence detection methods.
For instance, Cy5-coupled secondary antibodies were
shown only to be able to detect 6100 ng of antigen in
Western blots using a 450 nm blue LED (PhosphorImager; Molecular Dynamics, Sunnyvale, CA) while a
standard colorimetric nitroblue tetrazolium (NBT)/5bromo-4-chloro-3-indoyl phosphate (BCIP) system was
capable of detecting 3100 ng of antigen using alkaline
phosphatase-conjugated secondary antibody [164]. A
comparable commercialized detection system is the ECF
Western blotting system (Amersham Pharmacia Biotech),
which contains fluorescein-labeled secondary antibodies
for direct fluorescence detection of antigen. Sensitivity is
quite poor with this reagent, and it is only recommended
for detection of abundant proteins. Coupling alkaline
phosphatase-conjugated antifluorescein tertiary antibody
and the fluorogenic substrate AttoPhos to the direct fluorescence system results in a detection sensitivity that is
comparable to enhanced chemiluminescence detection,
albeit with additional incubation steps.
Phycobilisomes are highly organized, light-harvesting
antennae complexes attached to the stroma surface of
thylakoid or photosynthetic lamella in blue-green and red
algae. They are composed of several types of phycobiliproteins [165]. The phycobilisomes of red algae, for
example, consist of 84% phycoerythrin, 11% phycocyanin
and 5% allophycocyanin. All three proteins are composed
of a- and b-subunits and contain different isomeric linear
tetrapyrrole prosthetic groups attached through specific
cysteine residues by thioether linkages [165]. Isolated
1137
Figure 3. Schematic diagram

of PBXL-1 pigment (Red PBXL).
APC, allophycocyanin; PC, Rphycocyanin; PE, B-phycoerythrin. Energy transfer occurs as
indicated by the arrows. When
conjugated to a secondary antibody or streptavidin, the fluorescent pigment allows sensitive,
direct fluorescence detection of
targets on electroblot transfer
membranes.
phycobiliproteins have been used previously as labels in
immunoassays [166, 167] and as colored and fluorescent
molecular weight standards [165]. The recent introduction
of entire macromolecular phycobilisome assemblies as
reporter groups has boosted the performance of direct fluorescence-based immunodetection to sensitivity levels
that rival chemiluminescence procedures.
PBXL fluorescent pigments (Martek Biosciences, Columbia, MD) are chemically stabilized 1015 mega-Da phycobilisomes that contain 1400 fluorophores per complex
(Fig. 3) [168, 169]. The complexes, containing as many
as 45 phycobiliproteins, are stabilized by chemical crosslinking with agents such as glutaraldehyde and formaldehyde. The supramolecular structures allow direct fluorescent detection due to the large number of fluorophores
brought to each binding event. PBXL pigments are
roughly five orders of magnitude brighter than fluorescein
and three orders of magnitude brighter than cyanine dyes
(Cy3/Cy5) [170]. Visualization methods employing PBXL
pigments require fewer steps compared with typical
chemiluminescent approaches since no enzymatic amplification procedure is required. Thus, accessory substrates, chromogens, cofactors and timed incubations are
avoided [168]. Sub-pg detection sensitivity is often
achieved using PBXL pigments, with linear response
ranges encompassing 23 orders of magnitude. As little
as 0.78 pg of biotin-conjugated bovine serum albumin is
detectable using a streptavidin/PBXL-1(Red PBXL) conjugate while 0.5 ng of actin may be visualized using anti-
body-conjugated PBXL-3 (Crimson PBXL) [170]. PBXL

fluorescent pigments are characterized by large Stokes
shifts, and long wavelength emission (662666 nm) that
reduce interference from autofluorescence and permit
multicolor detection. PBXL-1 is spectrally well suited to
532 nm excitation sources such as the frequency doubled
Nd-YAG laser of the FMBIO II Fluorescence Imaging System (Hitachi, San Bruno, CA) or SHG laser source of the
FLA-3000G Fluorescence Image Analyzer (Fuji Photo
Film Co., Tokyo, Japan). The pigment may also be
excited with a 488 nm argon-ion laser, though emission
intensity decreases to roughly 36% of the maximum
obtainable output. PBXL-3 is maximally excited at 612 nm
and thus is appropriate for 633 nm He-Ne laser excitation
sources such as the laser employed in the FLA-3000G
Image Analyzer or 635 nm diode laser sources, such as
the one used with the Molecular Dynamics Storm operating in red fluorescence mode.
4.3 Fluorogenic alkaline phosphatase and

horseradish peroxidase substrates
Several attempts to utilize fluorogenic substrates for
detection of alkaline phosphatase- or horseradish peroxidase-conjugated antibodies have been reported in the literature. A potential advantage of this approach is that
unlike chemiluminescence, which generates light emission as a single event per molecule, cleavage of a fluorogenic substrate liberates a fluorescent product that can
be repetitively activated by the excitation beam and thus
1138
W. F. Patton
caused to fluoresce repeatedly. For Western blotting it is

possible to review results months or even years after an
experiment has been completed. Some examples of fluorogenic substrates are the peroxidase substrates 3-(4hydroxyphenyl)propionic acid and 2,7-dichlorodihydrofluorescein diacetate and the alkaline phosphatase substrates 4-methylumbelliferyl phosphate, 7-hydroxycoumarinyl phosphate, and fluorescein diphosphate [171
174]. Typically, substrates of this type produce watersoluble reaction products that can not effectively detect a
hybridization site on a membrane due to their diffusion.
Several fluorescent substrates for the detection of alkaline phosphatase or horseradish peroxidase that generate
a precipitation product have also been introduced [175
178]. The alkaline phosphatase substrate 3-hydroxy-N-2biphenyl-2-naphthalenecarboxamide phosphate ester
(HNPP)and the horseradish peroxidase substrate N(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB)
have been used for two-color fluorescence detection of
proteins electroblotted to nylon membranes [178]. Both
dyes may be excited using 302 nm UV illumination, with
HNPP producing a blue and AMMB producing a yellow
emission. Though PVDF membranes are generally used
for protein electroblotting, the AMMB reaction product is
not fluorescent on this substrate, possibly due to concentration quenching. The feasibility of a two-color fluorescence approach was evaluated using 128 ng of whole
serum proteins for the detection of two very prominent
components, serum albumin and IgG. Thus, detection
sensitivity appears to be quite poor using the method.
2-[2-Benzthiazoyl]-6-hydroxybenzthiazolyl
phosphate
(BBTP) is a sensitive substrate that is cleaved by alkaline
phosphatase to produce the highly fluorescent product
BBT (2-[2-benzthiazoyl]-6-hydroxybenzthiazole) [174].
BBTP is commercially available in pure form or as a component of the AttoPhos Plus Western Blot Kit (JBL Scientific, San Luis Obispo, CA). BBT is optimally excited at
420440 nm and maximally emits at 550560 nm. Linear
kinetics over a five log range of alkaline phosphatase
concentration has been demonstrated using AttoPhos
reagent in combination with certain imaging instruments
and detection sensitivity of 10 pg is reported by the manufacturer. Several other precipitable, fluorogenic substrates are commercially available that might also be
suitable for Western blotting. 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate; Molecular Probes) yields a hydrolysis product that is efficiently excited by the 633 nm spectral line of an He-Ne
laser and produces bright red fluorescence with emission
maximum of about 665 nm. Less than 1 ng of tubulin has
been detected using DDAO phosphate on Western blots
in combination with the Molecular Dynamics Storm operating in red fluorescence mode, according to the manu-

facturer. Additionally, ELF-97 phosphate (EnzymeLabeled Fluorescence; Molecular Probes) is an alkaline
phosphatase substrate with several unique properties
[177]. Upon enzymatic cleavage, this weakly blue-fluorescent substrate yields a bright yellow-green fluorescent
precipitate that exhibits an unusually large Stokes shift
and excellent photostability. The product is conveniently
excited by a 302 or 365 nm UV illumination source and
emits at about 520 nm. The spectral properties of ELF-97
phosphate are suitable for dichromatic protein detection
when used in combination with SYPRO Ruby Protein Blot
stain.
5 Fluorescence imaging devices

5.1 Overview of fluorescence detection
instrumentation
The primary role of analytical imaging systems is to
increase detection throughput in a reliable and convenient
manner, while generating results that are competitive
with nonimaging detection methods [156]. Fluorescence
images are typically recorded using a photographic camera, a CCD camera or a photomultiplier tube (PMT) [179,
180]. Film-based photography still remains integral to the
research enterprise, especially for recording protein profiles in laboratory notebooks and producing journal-quality
figures for scientific publications. Increasingly, bulk film
loaders, developing canisters, chemical developer and
fixer solutions are being supplanted by clean and convenient digital darkrooms with accompanying computer
graphics workstations. The instrumentation required for
conventional photography is not costly but the film and
processing costs are a recurring expense. While the spatial resolution obtained by film photography often surpasses that obtained with digital devices, the dynamic response of a photograph is limited to a 200-fold range
[179]. Quantitative analysis of fluorescently stained or
derivatized proteins is often performed using a CCD camera combined with a UV light box or xenon arc lamp. Another popular approach for imaging fluorescence is to use
a PMT detector combined with a laser light scanner.
5.2 CCD camera-based systems

Among the commonly available data acquisition devices,
CCD camera-based systems are the most versatile (Fig.
4). CCD camera-based imaging can be utilized for a variety of stained gels (CBB, silver, colloidal gold, fluorescently stained), autoradiographs, electroblots, thin-layer
chromatography (TLC) plates, multi-well plates and even
microarray biochips. CCD cameras are area imagers that
permit collection of fluorescence intensity through integration of the signal, often making bands visible that could
Figure 4. Components of a typical fixed CCD camera

imaging device. The Lumi-Imager F1 is shown in the diagram, though most other CCD systems have similar architectures. Diagram courtesy of Roche Molecular Biochemicals.
1139
Most CCD camera-based image analysis systems utilize

UV illumination to excite the fluorophores. It is expected
that in the near future, visible blue light illumination sources will become more common for the detection of fluorophores in proteomics, since they are less hazardous and
minimize photobleaching. A problem encountered with
gas-discharge light boxes in general is the lack of uniformity across the field of view. Many CCD camera systems perform mathematical flat field corrections within the
software to adjust for errors in shading arising from the
nonuniformity of the light source. An alternative light
source for CCD camera-based imaging systems is the
xenon arc lamp. High pressure xenon arc lamps provide
broad-band wavelength coverage and require modest
power. In combination with a very sensitive camera system, adequate excitation can be achieved to visualize
most fluorophores, and wavelength selection can be tailored using appropriate excitation and emission filters.
5.3 Laser scanner systems

not be detected by eye alone. 12-, 14- or 16-bit cooled
CCD cameras are commonly employed in gel image
acquisition and analysis systems. Theoretically, such systems should provide excellent quantitative information
over a range of 34 orders of magnitude. The image resolution obtained with currently implemented fixed CCD
cameras is usually inferior to photographic film. A number
of CCD chips are currently employed for gel imaging,
ranging in area from 1600 1200 to 640 480 pixels. A
typical 1024 1024 pixel CCD chip furnishes 200 mM resolution when large-format 20 20 cm gels are evaluated.
A small protein spot on a 2-D gel with 1 mm diameter
would thus be detected using about 1315 pixels. This
level of resolution is adequate and most 2-D analysis is
performed in this manner. However, improved resolution
is possible by mechanically scanning the CCD camera
over the sample and collecting multiple images that are
subsequently stitched back together to form a complete
image of the gel or blot. One such system, The Wallac
Arthur Multi-Wavelength Fluoroimager, delivers an amazing 50 mM resolution using this approach. There are tradeoffs associated with the higher resolution, however. Such
mechanical scanning systems are quite slow, with image
collection requiring as much as 30 min, as opposed to
310 s using fixed CCD camera systems. The X-Y movement of mechanical scanners requires a fairly large mechanism and systems based upon this design are big and
bulky compared with their fixed CCD camera counterparts. Finally, the integration of multiple images into a
composite may generate pictures characterized by a
patchwork pattern with varying background intensities in
each subregion.
Laser scanner-based systems serially pass an illumination beam over each point of the sample in a 2-D raster
pattern format [179]. This is achieved by optical scanning,
mechanical scanning, or a combination of the two approaches. Optical scanning uses a turning polygonal mirror, which reflects the incident light at various angles (Fig.
5). The incident light is passed through a lens and is then
reflected onto the sample. The lens adjusts the scanning
speed of the incident light between the center and edge of
the viewing field so that the center is not scanned faster
than the edges. It also adjusts the angle of the incident
light so that it is close to 90o over the entire image field.
Dual PMTs are often employed to allow simultaneous
detection of two fluorophores with concomitant increases
in throughput. Mechanical scanning physically moves the
laser scanner and PMT underneath the gel. Typically,
Figure 5. Components of a typical laser scanning device.

The FLA-3000G is shown in the diagram, though most
other laser systems have similar architecture. Diagram
courtesy of Dr. Kenji Miura, Fuji Photo Film, Tokyo,
Japan.
1140
W. F. Patton
Table 2. Summary of noncovalent fluorescent stains suitable for protein detection

Ex/Ema)
(nm)
Principal
applications
Features
300, 540/640
1-D gels
IEF gels
Lipoprotein detection
Blotting applications
Fair sensitivity (550 ng/band)

Similar performance as Coomassie
blue staining methods
SYPRO Ruby
protein gel stain
280, 450/610
2-D gels
Mass spectrometry
Edman sequencing
Highest sensitivity (12 ng/band)

Better performance than the best
silver staining methods
SYPRO Ruby IEF

protein gel stain
280, 450/610
IEF gels
Mass spectrometry
Edman sequencing
Highest sensitivity
Better performance than the best
silver staining methods
SYPRO Orange
protein gel stain
300, 470/570
1-D SDS-PAGE
Mass spectrometry
Edman sequencing
Capillary gel
electrophoresis
Good sensitivity (410 ng/band)

Little protein-to-protein variability
Better performance than colloidal
Coomassie blue staining methods
SYPRO Red
protein gel stain
300, 550/630
1-D SDS-PAGE
Mass spectrometry
Edman sequencing
Capillary gel
electrophoresis

Coomassie blue staining methods
SYPRO Tangerine
protein gel stain
300, 490/640
1-D SDS-PAGE
Blotting applications
Zymography
Electroelution
Mass spectrometry
Edman sequencing

No organic solvents or acids
Better performance than zincimidazole reverse staining methods
280, 450/618
Blotting membranesb)
Immunodetectionc)
Mass spectrometry
Edman sequencing

gold staining methods
SYPRO Rose Plus

protein blot stain
350d)/610
Immunodetectionc)
Mass spectrometry
Edman sequencing

Readily reversible
gold staining methods
SYPRO Rose
protein blot stain
350d)/590,
615
Immunodetectionc)
Mass spectrometry
Edman sequencing

Readily reversible
Better performance than Amido
Black staining methods
Dye name
Gel stains
Nile Red dye
Blot stains
SYPRO Ruby
protein blot stain
a)
b)
c)
d)
Ex, excitation maxima; Em, emission maxima

Nitrocellulose or PVDF membranes
i.e. Western blotting
UV epi-illumination only
optical scanning is much faster than mechanical X-Y

scanning, requiring as little as 6 min to interrogate an
entire 20 20 cm gel. While such laser scanners are still
substantially slower than a fixed CCD camera-based

imaging device, once again 50 mM resolution is achievable.

Though appropriate for the analysis of gels and electroblots, laser scanners lack the depth of focus to allow convenient analysis of multi-well plates. Another disadvantage of laser scanners is that they are limited to imaging
fluorophores that spectrally match the output of their laser
sources. Commonly employed sources in laser scanning
devices include the SHG laser (473 nm, 532 nm), Ar
laser (488 nm, 514 nm), frequency-doubled Nd-YAG
laser (532 nm), He-Ne laser (633 nm), and diode laser
(635 nm). Two or more laser sources are commonly
incorporated into commercial gel scanners, allowing a
wider number of fluorophores to be utilized. It is likely that
source wavelength constraints that currently restrict the
selection of fluorescence dyes will be relaxed somewhat
in the future as solid-state laser technology advances.
For example, commercial laser scanners do not currently
excite dyes with coherent UV illumination. While 365385
nm frequency-doubled alexandrite lasers are currently
expensive and unreliable, in the near future they may
become suitable for incorporation into laser gel scanning
devices. In the meantime, many UV excitable dyes used
to detect proteins are also readily excited with visible light
(Table 2).
6 Conclusions and perspectives

Correlating protein expression profiles with cell physiology requires rigorous determination of the quantity of
each protein in a sample. Unfortunately, the colorimetric
stains used for quantitatively evaluating protein expression are largely insufficient to the task (Fig. 1B). Though a
method to directly quantify proteins by mass spectrometry
without 2-D gels through covalent derivatization of cysteine residues with hydrogen and deuterium-containing
biotin affinity tags has recently been introduced, the linear
dynamic range of the method has explicitly been demonstrated over only a 4-fold range (Table 1; [181]). Additionally, ion suppression phenomenon associated with mass
spectrometry prevents stoichiometric comparison of different proteins by this technique. Current microchemical
methods used in proteome analysis require very low ng
levels of protein detection. Though silver staining procedures detect proteins in the low ng range, they are
cumbersome, multistep processes that exhibit quite poor
linear dynamic responses. Compatibility with mass spectrometry requires omission of glutaraldehyde from silver
stain formulations, which compromises staining performance [51]. An additional destaining step is also recommended to reduce background interference and suppression of signals often encountered in peptide mass
profiling experiments [41]. Fluorescence detection technologies offer greater sensitivity and broader linear
dynamic responses compared to their colorimetric counterparts.
1141
Preelectrophoretic covalent derivatization of proteins with

fluorophores invariably leads to altered protein mobility
[79, 80, 82]. This problem persists even when a single fluorophore is bound to a protein and could potentially lead
to misidentification of proteins excised from gels. Low
degreesof fluorophore substitution do permit analysis
of proteins by mass spectrometry and Edman-based
sequencing, but signals are quite dim and require sophisticated instruments for detection. Fluorophores that intercalate into the SDS micelles of proteins are fully compatible with modern proteomics investigations, but detection
sensitivity is not quite as good as the best available silver
staining methods [122126]. Such fluorophores are not
suitable for detecting proteins in IEF gels or on electroblot
membranes. Fluorophores that interact directly with proteins by a CBB-type mechanism, such as SYPRO Ruby
dye, are broadly compatible with modern proteomics
techniques and instrumentation [43, 50, 129]. Since the
dyes bind to proteins noncovalently after electrophoresis,
protein mobility shifts are avoided, permitting unambiguous excision of spots from gels. Peptides are unaltered by
the dyes, facilitating interpretation of mass profiling
results [43, 50, 126]. Though immunodetection remains
the domain of chemiluminescence detection technologies, PBXL pigments show potential for use with direct
fluorescence approaches [169]. PBXL pigments require
fewer steps compared with typical chemiluminescent approaches, since no enzymatic amplification procedure is
necessary. Sub-pg detection sensitivity is achievable and
samples may be evaluated years after the experiment
has been performed.
The future for fluorescence detection in proteomics
appears bright. New fluorescent staining methods, introduced only in the past two to three years, are finally delivering on the promise of superior performance advantages
over traditional colorimetric methods. As imaging instruments incorporate time-resolved capabilities for fluorescence detection, the sensitivity of such techniques are
likely to increase even further. An important advantage of
fluorescence detection is the ability to resolve two or more
fluorophores separately using optical filters. Increasingly,
it is expected that dichromatic and even polychromatic fluorescence detection strategies will find application in proteomics.
Received November 20, 1999
7 References
[1] Dunbar, T., Two-Dimensional Electrophoresis and Immunological Techniques, Plenum Press, New York, NY 1987,
p. 75.
[2] Gosling, J., Clin. Chem. 1990, 36, 14081427.
[3] Lopez, M., J. Chromatogr. B 1998, 722, 191202.
1142
W. F. Patton
[4] Fazekas de St. Groth, S., Webster, R., Datyner, A., Biochim. Biophys. Acta 1963, 71, 377391.
[34] Li, K., Geraerts, W., van Elk, R., Joose, J., Anal. Biochem.
1989, 182, 4447.
[5] Meyer, T., Lamberts, B., Biochim. Biophys. Acta 1965,

107, 144145.
[35] Moeremans, M., Daneels, G., De May, J., Anal. Biochem.

1985, 145, 315321.
[6] Merril, C., in: Chrambach, A., Dunn, M., Radola, B. J.

(Eds.) Advances in Electrophoresis, Vol. 1, VCH Publishers, New York 1987, pp. 111140.
[36] Moeremans, M., De Raeymaeker, M., Daneels, G., De

May, J., Anal. Biochem. 1986, 153, 1822.
[7] Wirth, P., Romano, A., J. Chromatogr. A 1995, 698,

123143.
[38] Schapira, A., Keir, G., Anal. Biochem. 1988, 169,

167171.
[8] Brush, M., The Scientist 1998, 12, 1622.
[39] Chevallet, M., Procaccio, V., Rabilloud, T., Anal. Biochem.

1997, 251, 6972.
[9] Diezel, W., Kopperschlager, G., Hofman, E., Anal. Biochem. 1972, 48, 617620.
[37] Egger, D., Bienz, K., Anal. Biochem. 1987, 166, 413417.
[10] Neuhoff, V., Stamm, R., Eibl, H., Electrophoresis 1985, 6,

427448.
[40] Velculescu, V., Zhang, L., Zhou, W., Vogelstein, J., Basrai,
M., Bassett, D., Hieter, P., Vogelstein, B., Kinzler, C., Cell
1997, 88, 243251.
[11] Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehrhardt,
W., Taube, D., Electrophoresis 1990, 11, 101117.
[41] Gharahdaghi, F., Weinberg, C., Meagher, D., Imai, B., Mische, S., Electrophoresis 1999, 20, 601605.
[12] Haebel, S., Albrecht, T., Sparbier, K., Walden, P., Korner,
R., Steup, M., Electrophoresis 1998, 19, 679686.
[42] Hunter, J., Hunter, S., Anal. Biochem. 1987, 164,

430433.
[13] Grassman, W., Hannig, K., Hoppe-Seylers Z. Physiol.

Chem. 1952, 290, 127.
[43] Berggren, K., Steinberg, T., Lauber, W., Carroll, J., Lopez,
M., Chernokalskaya, E., Zieske, L., Diwu, Z., Haugland,
R., Patton, W., Anal. Biochem. 1999, 276, 129143.
[14] Dunn, M., in: Link, A. (Ed.), Methods in Molecular Biology,

Humana Press, Totowa, NJ 1999, pp. 319329.
[15] Metkar, S., Mahajan, S., Sainis, J., Anal. Biochem. 1995,
227, 389391.
[16] Pryor, J., Xu, W., Hamilton, D., Anal. Biochem. 1992, 202,
100104.
[17] Eynard, L., Lauriere, M., Electrophoresis 1998, 19,
13941396.
[18] Johansson, K., Electrophoresis 1987, 8, 379383.
[19] Neumann, H., Mullner, S., Electrophoresis 1998, 19,
752757.
[20] Keranyi, L., Gallyas, F., Clin. Chem. Acta 1972, 38,
465467.
[21] Switzer, R., Merril, C., Shifrin, S., Anal. Biochem. 1979,
98, 231237.
[22] Rabilloud, T., in: Link, A. (Ed.), Methods in Molecular Biology, Humana Press, Totowa, NJ 1999, pp. 297305.
[23] Allen, R., Budowle, B., Protein Staining and Identification
Techniques, Eaton Publishing, Natick, MA 1999.
[24] Rabilloud, T., Vuillard, L., Gilly, C., Lawrence, J., Cell. Mol.
Biol. 1994, 40, 5775.
[25] Hager, D., Burgess, R., Anal. Biochem. 1980, 109, 7686.
[26] Lee, C., Levin, A., Branton, D., Anal. Biochem. 1987, 166,
308312.
[44] Li, K., Geraerts, W., van Elk, R., Joose, J., Anal. Biochem.
1988, 174, 97100.
[45] Miranda, P., Brandelli, A., Tezon, J., Anal. Biochem. 1993,
209, 376377.
[46] Gharahdaghi, F., Kirchner, M., Fernandez, J., Mische, S.,
Anal. Biochem. 1996, 233, 9499.
[47] Loo, R., Dales, N., Andrews, P., Prot. Sci. 1994, 11,
19751983.
[48] Christiansen, J., Houer, G., Electrophoresis 1992, 13,
179183.
[49] Quadroni, M., James, P., Electrophoresis 1999, 20,
664677.
[50] Berggren, K., Steinberg, T., Kemper, C., Lopez, M., Chernokalskaya, C., Diwu, Z., Haugland, R., Patton, W., Electrophoresis 2000, 21, in press.
[51] Shevchenko, A., Wilm, M., Vorm, O, Mann, M., Anal.
Chem. 1996, 68, 850858.
[52] Scheler, C., Lamer, S., Pan, Z., Li, X., Salnikow, J., Jungblut, P., Electrophoresis 1998, 19, 918927.
[53] Matsui, N., Smith-Beckerman, D., Epstein, L., in: Link, A.
(Ed.), Methods in Molecular Biology, Humana Press,
Totowa, NJ 1999, pp. 307312.
[54] Castellanos-Serra, L., Proenza, W., Huerta, V., Moritz, R.,
Simpson, R., Electrophoresis 1999, 20, 732737.
[27] Dzandu, J., Johnson, J., Wise, G., Anal. Biochem. 1988,
174, 157167.
[55] Castellanos-Serra, L., Fernandez-Patron, C., Hardy, E.,

Santana, H., Huerta, V. J., Prot. Chem. 1997, 16,
415419.
[28] Ortiz, M., Calero, M., Fernandez-Parton, C., Castellanos,

L., Mendez, E., FEBS Lett. 1992, 296, 300304.
[56] Talbot, D., Yphantis, D., Anal. Biochem. 1971, 44,

246253.
[29] Castellanos-Serra, L., Fernandez-Patron, C., Hardy, E.,

Huerta, V., Electrophoresis 1996, 17, 15641572.
[57] Weidekamm, E., Wallach, D., Fluckiger, R., Anal. Biochem. 1973, 54, 102114.
[30] Cohen, S., Chait, B., Anal. Biochem. 1997, 247, 257267.
[58] Ragland, W., Pace, J., Kemper, D., Anal. Biochem. 1974,
59, 2433.
[31] Hancock, K., Tsang, V., Anal. Biochem. 1983, 133,

157162.
[32] Rohringer, R., Holden, D., Anal. Biochem. 1985, 144,
118127.
[33] Falk, B., Elliott, C., Anal. Biochem. 1985, 144, 537541.
[59] Eng, P., Parkes, C., Anal. Biochem. 1974, 59, 323325.
[60] Stephens, R., Anal. Biochem. 1975, 65, 369379.
[61] Barger, B., White, F., Pace, J., Kemper, D., Ragland, W.,
Anal. Biochem. 1976, 70, 327335.
1143
[62] Carson, S., Anal. Biochem. 1977, 78, 428435.
[94] Alba, F., Daban, J., Electrophoresis 1997, 18, 19601966.
[63] Yamamato, K., Okamoto, Y., Sekine, T., Anal. Biochem.

1978, 84, 313318.
[95] Alba, F., Daban, J., Electrophoresis 1998, 19, 24072411.
[64] Schetters, H., McLeod, B., Anal. Biochem. 1979, 98,

329334.
[65] Araoz, R., Lebert, M., Hader, D., Electrophoresis 1998, 19,
215219.
[66] Chen-Kiang, S., Stein, S., Udenfriend, S., Anal. Biochem.
1979, 95, 122126.
[67] Jackowski, G., Liew, C., Anal. Biochem. 1980, 102,
321325.
[68] Alhanaty, E., Tauber-Finkelstein, M., Shaltiel, S., FEBS
Lett. 1981, 125, 151154.
[69] Taylor, M., Andrews, A., Electrophoresis 1981, 2, 7681.
[70] Tsugita, A., Sasada, S., van den Broek, R., Scheffler, J.,
Eur. J. Biochem. 1982, 124, 171176.
[96] Shultz, J., Leland, D., US Patent 5, 705, 649, 1998.

[97] Hermanson, G., Bioconjugate Techniques, Academic
Press, New York, NY 1996.
[98] Haugland, R., in: Spence, M. (Ed.), Handbook of Fluorescent Probes and Research Chemicals Molecular Probes
Inc., Eugene, OR 1996.
[99] Gygi, S., Rochon, Y., Franza, B., Aebersold, R., Mol. Cell.
Biol. 1999, 19, 17201730.
[100] OKeefe, D., Anal. Biochem. 1994, 222, 8694.
[101] Bravo, R., Celis, J., Clin. Chem. 1982, 28, 766781.
[102] Samso, M., Daban, J., Hansen, S., Jones, G., Eur. J. Biochem. 1995, 232, 818824.
[103] Shirahama, K., Tsujii, K., Takagi, T., J. Biochem. (Tokyo)
1974, 75, 309319.
[71] Strottman, J., Robinson, J., Stellwagen, E., Anal. Biochem.

1983, 132, 334337.
[104] Stellwagen, N., Electrophoresis 1998, 19, 15421547.
[72] Law, H., Lingwood, C., Anal. Biochem. 1985, 149,

404408.
[105] Holmes, D., Stellwagen, N., Electrophoresis 1991, 12,

612619.
[73] Vandekerckhove, J., Bawn, G., Puype, M., Van Damme,

J., Van Montagu, M., Eur. J. Biochem. 1985, 152, 919.
[106] Righetti, P. G., Caglio, S., Saracchi, M., Quaroni, S., Electrophoresis 1992, 13, 587595.
[74] Summers, D., Szewczyk, B., in: Walker, J. (Ed.), The Protein Protocols Handbook Humana Press, Totowa, NJ
1996, pp. 289301.
[107] Ruchel, R., Steere, R., Erbe, E., J. Chromatogr. 1978,

166, 563575.
[75] Vera, J., Rivas, C., Anal. Biochem. 1988, 173, 399404.
[76] Jackson, P., US Patent 5, 320, 727, 1994.
[77] Crawford, N., Droux, M., Kosower, N., Buchanan, B., Arch.
Biochem. Biophys. 1989, 271, 223239.
[78] Houston, B., Peddie, D., Anal. Biochem. 1989, 177,
263267.
[79] Urwin, V., Jackson, P., Anal. Biochem. 1993, 209, 5762.
[80] Unlu, M., Morgan, M., Minden, J., Electrophoresis 1997,
18, 20712077.
[81] Tejero-Diez, P., Rodriguez-Sanchez, P., Diez-Guerra, F.,
Anal. Biochem. 1999, 274, 278282.
[82] Jackson, P., Urwin, V., Mackay, C., Electrophoresis 1988,
9, 330339.
[108] Matulis, D., Lovrien, R., Biophys. J. 1998, 74, 422429.

[109] Matulis, D., Richardson, T., Lovrien, R., J. Mol. Recognit.
1996, 9, 433443.
[110] Conroy, M., Lovrien, R., J. Cryst. Growth 1992, 122,
213222.
[111] Daban, J., Bartolome, S., Samso, M., Anal. Biochem.
1991, 199, 169174.
[112] Wilkins, M., Gasteiger, E., Sanchez, J.-C., Bairoch, A.,
Hochstrasser, D., Electrophoresis 1998, 19, 15011505.
[113] de Jong, W., Zweers, A., Cohen, L., Biochem. Biophys.
Res. Commun. 1978, 82, 532539.
[114] Hartman, B., Udenfriend, S., Anal. Biochem. 1969, 30,
391394.
[83] Urwin, V., Jackson, P., Anal. Biochem. 1991, 195, 3037.
[115] Nerenberg, S., Ganger, C., DeMarco, L., Anal. Biochem.

1971, 43, 564574.
[84] Kosower, E., Kosower, N., Methods Enzymol. 1995, 251,

133148.
[116] Daban, J., Aragay, A., Anal. Biochem. 1984, 138,

223228.
[85] Kosower, N., Kosower, E., Newton, G., Ranney, H., Proc.
Natl. Acad. Sci. USA 1979, 76, 33823386.
[117] Pina, B., Aragay, A., Suau, P., Daban, J., Anal. Biochem.
1985, 146, 431433.
[86] Kosower, N., Newton, G., Kosower, E., Ranney, H., Biochim. Biophys. Acta 1980, 622, 201209.
[118] Daban, J., Bartolome, S., Bermudez, A., in: Walker, J.

(Ed.), The Protein Protocols Handbook Humana Press,
Totowa, NJ 1996, pp. 179185.
[87] Kosower, N., Zipser, Y., Faltin, Z., Biochim. Biophys. Acta
1982, 691, 345352.
[88] Shalgi, R., Seligman, J., Kosower, N., Biol. Reprod. 1989,
40, 10371045.
[119] Horowitz, P., Bowman, S., Anal. Biochem. 1987, 165,

430434.
[89] Cornwall, G., Chang, T., J. Androl. 1990, 11, 168181.
[120] Daban, J., Samso, M., Bartolome, S., Anal. Biochem.

1991, 199, 162168.
[90] Huang, T., Kosower, N., Yanagimachi, R., Biol. Reprod.

1984, 31, 797809.
[121] Bermudez, A., Daban, J., Garcia, J., Mendez, E., BioTechniques 1994, 16, 621624.
[91] Seligman, J., Kosower, N., Weissenberg, R., Shalgi, R.,

J. Reprod. Fertil. 1994, 101, 435443.
[122] Steinberg, T., Jones, L., Haugland, R., Singer, V., Anal.
Biochem. 1996, 239, 223237.
[92] Fey, S., Nawrocki, A., Larsen, M., Grg, A., Roepstorff, P.,
Skews, G., Williams, R., Larsen, P., Electrophoresis 1997,
18, 13611372.
[123] Steinberg, T., Haugland, R., Singer, V., Anal. Biochem.

1996, 239, 238245.
[93] Goldberg, R., Fuller, G., Anal. Biochem. 1979, 90, 6980.
[124] Steinberg, T., White, H., Singer, V., Anal. Biochem. 1997,
248, 168172.
1144
W. F. Patton
[125] Haugland, R., Singer, V., Jones, L., Steinberg, T., US Patent 5, 616, 502, 1997.
[154] Deo, S., Fule, R., Saoji, A., Indian J. Exp. Biol. 1990, 28,
11841186.
[126] Steinberg, T., Lauber, W., Berggren, K., Kemper, C., Yue,
S., Patton, W., Electrophoresis 1999, 20, in Press.
[155] Tsang, V., Hancock, K., Simons, A., Anal. Biochem. 1984,
143, 304307.
[127] Lim, M., Patton, W., Shojaee, N., Lopez, M., Spofford, K.,
Shepro, D., Anal. Biochem. 1997, 245, 184195.
[128] Patton, W., Lim, M., Shepro, D., in: Link A. (Ed.), Methods
in Molecular Biology, Humana Press, Totowa, NJ 1999,
pp. 331339.
[129] Steinberg, T., Chernokalskaya, E., Berggren, K., Lopez,
M., Diwu, Z., Haugland, R., Patton, W., Electrophoresis
1999, 20, in press.
[130] Fowler, S., Brown, W., Warfel, J., Greenspan, P., J. Lipid
Res. 1987, 28, 12251232.
[131] Spiekermann, P., Rehm, B., Kalscheuer, R., Baumeister,
D., Steinbuchel, A., Arch. Microbiol. 1999, 171, 7380.
[132] Gorenflo, V., Steinbuchel, A., Marose, S., Rieseberg, M.,
Scheper, T., Appl. Microbiol. Biotechnol. 1999, 51,
765772.
[133] Takagi, T., Kubo, K., Isemura, T., Biochim. Biophys. Acta
1980, 623, 271279.
[134] Alba, F., Bermudez, A., Bartolome, S., Daban, J., BioTechniques 1996, 21, 625626.
[135] Sackett, D., Wolff, J., Anal. Biochem. 1987, 167, 228234.
[136] Greenspan, P., Gutman, R., Electrophoresis 1993, 14,
6568.
[137] Greenspan, P., Mayer, E., Fowler, S., J. Cell Biol. 1985,
100, 965973.
[138] Greenspan, P., Mao, F., Ryu, B., Gutman, R., J. Chromatogr. A 1995, 698, 333339.
[139] Harvey, M., Bandilla, D., Banks, P., Electrophoresis 1998,
19, 21692174.
[140] Hamby, R., Am Biotechnol. Lab. 1996, 14, 12.
[141] Hamby, R., Biotechnol. Int. 1997, 1, 339343.
[142] Patton, W., Lim, M., Shepro, D., in: Link, A. (Ed.), Methods
in Molecular Biology, Humana Press, Totowa, NJ 1999,
pp. 353362.
[143] Graham, G., Nairn, R., Bates, G., Anal. Biochem. 1978,
88, 434441.
[144] Bickar, D., Reid, P., Anal. Biochem. 1992, 203, 109115.
[145] Patton, W., Lam, L., Su, Q., Lui, M., Erdjument-Bromage,
H., Tempst, P., Anal. Biochem. 1994, 220, 324335.
[146] Shojaee, N., Patton, W., Lim, M., Shepro, D., Electrophoresis 1996, 17, 687693.
[147] Lim, M., Patton, W., Shojaee, N., Shepro, D., BioTechniques 1996, 21, 888897.
[148] Griffith, I., Anal. Biochem. 1972, 48, 402412.
[149] Sun, S., Hall, T., Anal. Biochem. 1974, 61, 237242.
[150] Bosshard, H., Datyner, A., Anal. Biochem. 1977, 82,
327333.
[151] Saoji, A., Jad, C., Kelkar, S., Clin. Chem. 1983, 29, 4244.
[152] Saoji, A., Jad, C., Yemul, V., Khare, P., Kelkar, S., Clin.
Chem. 1984, 30, 12521254.
[153] Tzeng, M., Anal. Biochem. 1983, 128, 412414.
[156] Ramm, P., Drug Discovery Today 1999, 4, 401410.

[157] Krika, L., Thorpe, G., Whitehead, T., US Patent 4, 598,
044, 1986.
[158] Thorpe, G., Krika, L., Moseley, M., Whitehead, T., Clin.
Chem. 1985, 31, 13351341.
[159] Guy, G., in: Walker, J. (Ed.), The Protein Protocols Handbook Humana Press, Totowa, NJ 1996, pp. 329335.
[160] Paladichuk, A., The Scientist 1999, 13, 18.
[161] Bronstein, I., Voyta, J., Murphy, O., Bresnick, L., Kricka,
L., BioTechniques 1992, 12, 748753.
[162] Schaap, A., Bronstein, I., US Patent 5,707,559, 1998.
[163] Bronstein, I., Edwards, B., Sparks, A., US Patent
5,869,699, 1999.
[164] Fradelizi, J., Friederich, E., Beckerle, M., Golsteyn, R.,
BioTechniques 1999, 26, 484494.
[165] Glazer, A., Methods Enzymol. 1988, 167, 291303.
[166] Stryer, L., Glazer, A., Oi, V., US Patent 4,520,110, 1985.
[167] Kronick, M., Grossman, P., Clin. Chem. 1983, 29,
15821586.
[168] Cubicciotti, R., US Patent 5,695,990, 1997.
[169] Morseman, J., Moss, M., Zoha, S., Allnutt, F., BioTechniques 1999, 26, 559563.
[170] Zoha, S., International Business Communications Conference on Detection Technologies; Applications in Fluorescence and Probe Technologies for Drug Discovery and
Clinical Diagnosis, Sheraton Hotel and Towers, Seattle,
WA, June 2425, 1999.
[171] Ferrer, A., Santema, J., Hilhorst, R., Visser, A., Anal. Biochem. 1990, 187, 129132.
[172] Zaitsu, K., Ohkura, Y., Anal. Biochem. 1980, 109,
109113.
[173] Manchenko, G., Handbook of Detection of Enzymes on
Electrophoretic Gels, CRC Press, New York, NY 1994, pp.
187191.
[174] Klem, R., Marvin, W., US Patent 5,424,440, 1995.
[175] Cano, R., Torres, M., Klem, R., Palomares, J., BioTechniques 1992, 12, 264269.
[176] Kagiyama, N., Fujita, S., Momiyama, M., Saito, H., Shirahama, H., Hori, S., Acta Histochem. Cytochem. 1992, 25,
467471.
[177] Larison, K., BreMiller, R., Wells, K., Clements, I., Haugland, R., J. Histochem. Cytochem. 1995, 43, 7783.
[178] Kondoh, Y., Fujita, S., Kagiyama, N., Yoshida, M., DNA
Res. 1998, 5, 217220.
[179] Sutherland, J., in: Chrambach, A., Dunn, M., Radola, B. J.
(Eds.), Advances in Electrophoresis, Vol. 6 VCH Publishers, New York 1993, pp. 142.
[180] Patton, W., J. Chromatogr. A 1995, 698, 5587.
[181] Gygi, S., Rist, B., Gerber, S., Turecek, F., Gelb, M., Aebersold, R., Nature Biotechnol. 1999, 17, 994999.

A Thousand Points of Light The Application of Fluo

Transféré par

Informations du document

Titre original

Copyright

Formats disponibles

Partager ce document

Partager ou intégrer le document

Options de partage

Avez-vous trouvé ce document utile ?

Ce contenu est-il inapproprié ?

Droits d'auteur :

Formats disponibles

A Thousand Points of Light The Application of Fluo

Transféré par

Droits d'auteur :

Formats disponibles

Electrophoresis 2000, 21, 11231144