New fluorescent stains for protein detection in sodium dodecyl sulfate-polyacrylamide gels

Article abstract of DOI:10.1246/cl.2004.318

Highly sensitive new fluorescent stains for proteins in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) were developed by combining an environment sensitive fluorophore and a hydrocarbon tail for improved binding to SDS molecules associated with the proteins. The experimental results showed that these dyes bound to protein-SDS complexes with a concomitant emission increase upon binding.

Full text of DOI:10.1246/cl.2004.318

318
Chemistry Letters Vol.33, No.3 (2004)
New Fluorescent Stains for Protein Detection in Sodium Dodecyl Sulfate–Polyacrylamide Gels
Soo Yeon Hong, Hyunsook Jun, Seung Soo Yoon,ꢀ Chulhun Kang,y and Myungkoo Suhꢀ
Department of Chemistry, Sung Kyun Kwan University, Suwon 440-746, Korea
yDepartment of Neuroscience, Graduate School of East-West Medical School, Kyung Hee University,
Yongin 449-701, Korea
(Received November 21, 2003; CL-031130)
Highly sensitive new fluorescent stains for proteins in so-
dium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) were
developed by combining an environment sensitive fluorophore
and a hydrocarbon tail for improved binding to SDS molecules
associated with the proteins. The experimental results showed
that these dyes bound to protein–SDS complexes with a concom-
itant emission increase upon binding.
H3C
R
H3C
R
O
H3C
H
POCl3
DMF
R X
Cs2CO3/DMF
N
N
N
H
EtOH
O
N
S
N
Se
N
Fluorescent dyes are increasingly used for visualization of
proteins in sodium dodecyl sulfate–polyacrylamide gels (SDS–
PAGE) due to their high sensitivity and ease of use.1 Unlike co-
valent modifiers, fluorescent stains which bind non-covalently to
proteins or SDS molecules associated with proteins are particu-
larly useful for proteomic studies.2 These non-covalent binders
do not interfere post-gel microanalysis of the visualized proteins
by means of mass spectrometry, which is essential for protein
identification nowadays.
Various environment-sensitive fluorescent dyes were report-
ed to increase emission intensity in the presence of hydrophobic
protein–SDS complexes, and used as a protein detection reagent
in gels and/or in solutions. A hydrophobic probe, 1-anilino-
naphthalene-8-sulfonate (ANS) was first applied for protein
staining3 and later Nile red was used as a protein detection re-
agent for SDS–PAGE and for solutions with much improved
sensitivity.4 More recently, new dyes, SYPRO1 Orange, Red,
and Tangerine, which exhibit intense fluorescence upon binding
to protein–SDS complexes, were developed as protein stains for
SDS–PAGE.5 These dyes can visualize protein bands which con-
tain as low as 4–10 nanogram proteins.
O
S
Se
+
+
+
N
N
N
N
N
N
H C
H C
H C
3
3
3
R
R
R
3d, R = C15H31
2a, R = C12H13
1d, R = C15H31
2b,
2c,
2d,
2e,
C13H17
C14H29
C15H31
C18H37
Scheme 1. Structures and synthesis of fluorescent stains.
ex
em
2
1
3
In the present study, we developed new fluorescent dyes,
which are to be used as sensitive non-covalent fluorescent stains
for proteins in SDS–PAGE. The synthesized stains (1, 2, and 3 in
Scheme 1) contain a medium-sensitive fluorophore, which has
an electron accepting group and an electron donating amino
group, and a long hydrocarbon tail for increasing affinity to
SDS molecules associated with proteins.6 Either benzoxazoli-
um-, benzothiazolium-, or benzoselenazolium-group was em-
ployed as an electron accepting group. In order to investigate
the effect of hydrocarbon tail, the tail length was also varied.
The fluorescent stains were synthesized according to
Scheme 1. Knoevenagel reactions of 4-(methyl-alkyl-amino)-
benzaldehyde with 2,3-dimethylbenzoxazol-3-ium (1), 2,3-di-
methylbenzothiazol-3-ium (2), or 2,3-dimethylbenzoselen-
azol-3-ium (3) were carried under refluxing condition in ethanol.
Molecular structures of the fluorescent probes were confirmed
by their spectroscopic data.7
400
500
600
700
Wavelength /nm
Figure 1. Normalized fluorescence excitation and emission spectra
of the synthesized stains in ethanol. Dashed lines, solid lines and
dotted lines represent 1d, 2d, and 3d, respectively.
of benzooxazolium derivative (1) is about 50 nm blue-shifted
compared to benzothiazolium (2) or benzoselenazolium deriva-
tives (3). Nevertheless, all synthesized dyes can be excited effi-
ciently by using commercially available light sources in gel
scanners, such as 488 or 532-nm laser lights. Absorption and
emission characteristics of the dyes in ethanol are summarized
in Table 1.
As shown in Figure 1, the excitation maxima of the synthe-
sized dyes are in the range of 500–550 nm. Both excitation and
emission wavelengths increase in the order of 1, 2, and 3. Spectra
The quantum yields of the dyes in aqueous solutions are ex-
ceptionally low, making the dyes virtually non-fluorescent.
Copyright ꢀ 2004 The Chemical Society of Japan

Chemistry Letters Vol.33, No.3 (2004)
319
Table 1. Fluorescence characteristics of the synthesized stains
ꢁabs
/nm
ꢁem
/nm
Enhancement
factorb
Stain
ꢂa
1d
2a
2b
2c
2d
2e
3d
500
532
531
534
534
538
544
555
595
596
594
596
590
599
0.037
0.013
0.019
0.020
0.024
0.021
0.023
110
90
40
50
170
280
366
Figure 3. SDS–PAGE gel images stained with the synthesized
dyes. Molecular weight marker proteins were loaded by twofold se-
rial dilution starting from 100 ng/band at the most left lane.
Fluorescence spectra are measured with 1.0 mM dye solutions in
sensitivity. Protein bands containing less than 1 ng proteins (8th
lane) were readily visualized and the sensitivity was maintained
after prolonged exposure to the laser light by scanning the gel re-
peatedly (>5 times attempted).
In conclusion, fluorescent dyes consisting of an environment
sensitive fluorophore and a hydrocarbon tail can be excellent
non-covalent protein stains for SDS–PAGE. Further effort for
developing sensitive protein stains is underway in our group.
a
b
ethanol. Fluorescence quantum yields. Fluorescence emission
increase in the presence of 150 mg/mL BSA and 0.05% SDS
compared to the one in the presence of 0.05% SDS only in aque-
ous 1.5% acetic acid solution.
However, the emission increased greatly in the presence of pro-
teins exhibiting intense fluorescence in aqueous solutions. This
drastic change of fluorescence spectra is demonstrated in
Figure 2. It should be noted that this emission increase was ob-
served only when both BSA and SDS are present, which
indicates that the dyes interact with SDS molecules associated
with proteins. The quantum yields of the dyes in BSA solution
increased gradually with increasing BSA concentration, and then
reached ꢁ0:1. The extent of enhancement increased in the order
of 1d, 2d, and 3d (Table 1). Also the length of hydrocarbon tail
affected the emission increase. We found that this emission in-
crease is largely attributed to the increase of fluorescence life-
time (inset of Figure 2). Binding of the dyes to BSA–SDS com-
plex restricts mobility of the dyes and reduces nonradiative
relaxation of the excited dye molecules, which in turn increases
the fluorescence lifetime.
We attempted to visualize molecular weight marker proteins
in SDS–PAGE by staining with the synthesized dyes. After the
electrophoresis was completed, the gel was fixed followed by in-
cubation in the staining solution containing 1 mM dye and 5%
ethanol for 1 h. The resulting gel images (Figure 3) obtained
by irradiating the gels with 532-nm light indicate that all synthe-
sized dyes successfully stained proteins in SDS–PAGE with high
We thank Dr. Freek Ariese for his help with fluorescence
lifetime measurements. This work was supported by a program
(R05-2003-000-11419-0) of Korea Science and Engineering
Foundation and the Brain Korea 21 Program of the Ministry of
Education, Korea.
References and Notes
1
2
3
W. F. Patton, J. Chromatogr., B, 771, 3 (2002) and references therein.
W. F. Patton, Electrophoresis, 21, 1123 (2000).
a) B. K. Harman and S. Udenfriend, Anal. Biochem., 30, 391 (1969). b) J.
R. Daban and A. M. Aragay, Anal. Biochem., 138, 223 (1984).
a) J. R. Daban, S. Bartolome, and M. Samso, Anal. Biochem., 199, 169
(1991). b) J. R. Daban, M. Samso, and S. Bartolome, Anal. Biochem.,
199, 162 (1991).
a) T. H. Steinberg, L. J. Jones, R. P. Haugland, and V. L. Singer, Anal.
Biochem., 239, 223 (1996). b) T. H. Steinberg, W. M. Lauber, K.
Berggren, C. Kemper, S. Yue, and W. F. Patton, Electrophoresis, 21,
497 (2000).
4
5
6
7
C. Kang, H. J. Kim, D. Kang, D. Y. Jung, and M. Suh, Electrophoresis, 24,
3297 (2003).
Selected data for 1d, 1H NMR (CDCl3, 500 MHz) ꢀ 0.88 (t, 3H), 1.10–
1.38 (m, 24H), 1.61 (t, 2H), 3.07 (s, 3H), 3.39 (t, 2H), 4.37 (s, 3H), 6.68
(d, 2H), 7.52 (d, 1H), 7.54 (d, 1H), 7.57 (d, 1H), 7.67 (t, 2H), 8.00 (d,
2H), 8.14 (d, 1H), HRMS (FABþ) m=z calcd for C32H47N2Oþ,
475.3688 (Mþ) observed, 475.3688; 2a, 1H NMR (CDCl3, 500 MHz) ꢀ
0.88 (t, 3H), 1.20–1.38 (m, 18H), 1.55 (t, 2H), 2.95 (s, 3H), 3.28 (t,
2H), 4.33 (s, 3H), 6.51 (d, 2H), 7.45 (td, 1H), 7.54–7.60 (m, 2H), 7.64
(d, 1H), 7.86 (d, 1H), 7.94 (d, 2H), 8.08 (d, 1H), HRMS (FABþ) m=z calcd
for C29H41N2Sþ, 449.2990 (Mþ) observed, 449.2995; 2b, 1H NMR
(CDCl3, 500 MHz) ꢀ 0.88 (t, 3H), 1.17–1.36 (m, 20H), 1.61 (t, 2H),
3.05 (s, 3H), 3.39 (t, 2H), 4.44 (s, 3H), 6.67 (d, 2H), 7.54 (td, 1H), 7.64
(td, 1H), 7.67 (d, 1H), 7.78 (d, 1H), 7.83 (d, 1H), 7.95–8.05 (m, 3H),
HRMS (FABþ) m=z calcd for C30H43N2Sþ, 463.3147 (Mþ) observed,
463.3159; 2c, 1H NMR (CDCl3, 500 MHz) ꢀ 0.88 (t, 3H), 1.16–1.37 (m,
22H), 1.59 (t, 2H), 3.02 (s, 3H), 3.56 (t, 2H), 4.40 (s, 3H), 6.63 (d, 2H),
7.53 (td, 1H), 7.61 (td, 1H), 7.64 (d, 1H), 7.75 (d, 1H), 7.85 (d, 1H),
7.98 (d, 2H), 8.00 (d, 1H), HRMS (FABþ) m=z calcd for C31H45N2Sþ,
477.3303 (Mþ) observed, 477.3306; 2d, 1H NMR (CDCl3, 500 MHz) ꢀ
0.88 (t, 3H), 1.21–1.35 (m, 26H), 1.61 (t, 2H), 3.01 (s, 3H), 3.38 (t,
2H), 4.43 (s, 3H), 6.66 (d, 2H), 7.55 (td, 1H), 7.64 (td, 1H), 7.67 (d,
1H), 7.78 (d, 1H), 7.83 (d, 1H), 7.92–8.00 (m, 3H), HRMS (FABþ) m=z
calcd for C32H47N2Sþ, 491.3460 (Mþ) observed, 491.3447; 2e, 1H
NMR (CDCl3, 500 MHz) ꢀ 0.88 (t, 3H), 1.18–1.35 (m, 30H), 1.59 (t,
2H), 3.03 (s, 3H), 3.36 (t, 2H), 4.41 (s, 3H), 6.64 (d, 2H), 7.53 (td, 1H),
7.63 (td, 1H), 7.65 (d, 1H), 7.75 (d, 1H), 7.84 (d, 1H), 7.96 (d, 2H),
8.00 (d, 1H), HRMS (FABþ) m=z calcd for C35H53N2Sþ, 533.3929
(Mþ) observed, 533.3914; 3d, 1H NMR (CDCl3, 500 MHz) ꢀ 0.88 (t,
3H), 1.12–1.38 (m, 22H), 1.61 (t, 2H), 2.02 (t, 2H), 3.03 (s, 3H), 3.36
(t, 2H), 4.26 (s, 3H), 6.60 (d, 2H), 7.42 (td, 1H), 7.49–7.55 (m, 2H),
7.59 (d, 1H), 7.70 (d, 1H), 7.87 (d, 2H), 8.30 (d, 1H), HRMS (FABþ)
m=z calcd for C32H47N2Seþ, 539.2904 (Mþ) observed, 539.2921.
12
40
10
8
6
30
0
5
10
15
Time /nanoseconds
20
10
0
400
500
600
700
800
Wavelength /nm
Figure 2. Fluorescence enhancement of 2a in the presence of pro-
teins. Solid lines represent excitation and emission fluorescence
spectra in the presence of 150 mg/mL BSA and 0.05% SDS. Dotted
lines are for the ones in the presence of 0.05% SDS only. Inset de-
picts the fluorescence lifetimes of 2a in the presence (ꢃav ¼ 1:1 ns,
solid line) and absence of BSA (ꢃav < 30 ps, dotted line). All solu-
tions contain 1.5% acetic acid. ½2aꢂ ¼ 1 mM.
Published on the web (Advance View) February 14, 2004; DOI 10.1246/cl.2004.318