Angewandte
Chemie
Fluorescent Probes
analysis highly desirable. Herein, we report a new probe for
ALP based on a fluorescein derivative that we developed
recently which works effectively even on Western blots and
makes it possible to perform multicolor labeling.
A critical requirement of probes for Western blot analysis
is that the hydrolysis product has adequate affinity for the
blot. We confirmed that fluorescein generated by ALP-
catalyzed hydrolysis of FDP, lacks sufficient affinity for the
blocked membrane in Western blotting (Figure 1), and we
[
18]
DOI: 10.1002/anie.200501542
Extension of the Applicable Range of
Fluorescein: A Fluorescein-Based Probe for
Western Blot Analysis**
Mako Kamiya, Yasuteru Urano, Nobuyoshi Ebata,
Masami Yamamoto, Jyunichi Kosuge, and
Tetsuo Nagano*
Western blot immunoanalysis is a powerful method for
detecting proteins of interest on a nitrocellulose or polyviny-
[
1–3]
lidene fluoride (PVDF) membrane.
The presence of a
target protein is usually detected by using a secondary
antibody conjugated with an enzyme such as alkaline
phosphatase (ALP), which allows marked signal amplifica-
[
4]
tion, in combination with a chromogenic, fluorescent, or
[
5–7]
chemiluminescent probe.
fluorescence detection offers high sensitivity and the possi-
Among these techniques, only
Figure 1. Difference in affinity for the blot between 2-Me-4-OMe TG
and fluorescein. Solutions of various concentrations (100 mm, 10 mm,
[
8,9]
bility of multicolor labeling.
Furthermore, fluorescence
1
mm, 100 nm) of 2-Me-4-OMe TG (right lane) and fluorescein (left
signals, unlike transient chemiluminescence signals, can be
imaged several times and are stable on a dried blot. However,
the range of fluorescence probes applicable to Western blot
analysis is limited at the present time. In particular, probes
based on the fluorescein molecule have rarely been used in
this analysis despite their extensive exploitation in other
lane) were dropped onto a blocked nitrocellulose membrane and
imaged a) before and b) after washing.
suggest that this low affinity may be attributed to the
carboxylic group of the fluorescein molecule which makes
the hydrophilicity rather high. We then examined the affinity
of 2-Me TG, one of our recently reported fluorescein
[
10–13]
biological techniques.
For example, fluorescein diphos-
[
14–17]
phate (FDP),
a fluorescein-based ALP probe, is seldom
[
18]
used to detect the activity of the widely used secondary
detection enzyme ALP in Western blot analysis. Nevertheless,
the superior optical properties of fluorescein, such as high
molar extinction coefficients and high quantum yields of
fluorescence in aqueous media as well as efficient excitation
by an argon ion laser (488 nm), make the development of
fluorescein-based probes for ALP suitable for Western blot
derivatives (TokyoGreens, TG),
in which the carboxylic
group of the traditional fluorescein molecule is replaced with
a methyl group. We found that a spot of a solution of 2-Me TG
dropped onto the membrane remained well defined and was
not readily washed out (see Supporting Information). This
result suggested that TokyoGreens, which do not have a
carboxylic group, are more suitable than traditional fluores-
cein as the fluorophore in probes for Western blot analysis.
TokyoGreens also offer the advantage of precise control of
the fluorescence emission output. We thus considered 2-Me-
[
+]
[
*] M. Kamiya, Dr. Y. Urano, Prof. Dr. T. Nagano
Graduate School of Pharmaceutical Sciences
The University of Tokyo
4
-OMe TG (Figure 1), which shows strong fluorescence (as
fluorescein does) and becomes essentially nonfluorescent
upon alkylation of its phenolic group, as a suitable core
fluorophore for our new probe. Incorporation of a phosphate
group into the phenolic group of 2-Me-4-OMe TG gave TG-
Phos, a new probe for ALP whose hydrolysis product has a
high affinity for Western blots, unlike the hydrolysis product
of FDP (Scheme 1).
TG-Phos is stable and almost nonfluorescent, but is
converted into strongly fluorescent 2-Me-4-OMe TG upon
one-step hydrolysis by ALP (see Scheme 1a, Figure 2a, and
Supporting Information). We observed a linear relationship
between ALP activity and the initial rate of increase of
fluorescence intensity (Figure 2b). In the case of FDP, which
is hydrolyzed first to moderately fluorescent fluorescein
monophosphate (FMP) and then to strongly fluorescent
fluorescein, the rate of increase of fluorescence is slow
(Scheme 1b, Figure 2a). Additionally, there is poor linearity
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax: (+81)3-5841-4855
E-mail: tlong@mol.f.u-tokyo.ac.jp
N. Ebata, M. Yamamoto, J. Kosuge
Development Department, Research Products Division
Daiichi Pure ChemicalsCo., Ltd.
3
-3-1 Koyodai, Ryugasaki-city, Ibaraki 301-0852 (Japan)
+
[
] PRESTO, Japan Science and Technology Agency
Kawaguchi, Saitama (Japan)
[
**] This study was supported in part by a grant for Precursory Research
for Embryonic Sciencesand Technology from the JSTAgency to Y.U.,
aswell asre se arch grants(grant no s. 13557209, 16651106, and
16689002) to Y.U. and a grant for the Advanced and Innovational
Research Program in Life Sciences to T.N. from the Ministry of
Education, Culture, Sports, Science, and Technology of the Japanese
Government.
Supporting information for thisarticle isavailable on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 5439 –5441
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5439