A rapid SNAP-tag fluorogenic probe based on an environment-sensitive fluorophore for no-wash live cell imaging

Article abstract of DOI:10.1021/cb500502n

(Figure Presented). One major limitation of labeling proteins with synthetic fluorophores is the high fluorescence background, which necessitates extensive washing steps to remove unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on an environment-sensitive fluorophore for labeling with SNAP-tag proteins. The probe exhibits dramatic fluorescence turn-on of 280-fold upon being labeled to SNAP-tag. The major advantages of our fluorogenic probe are the dramatic fluorescence turn-on, ease of synthesis, high selectivity, and rapid labeling with SNAP-tag. No-wash labeling of both intracellular and cell surface proteins was successfully achieved in living cells, and the localization of these proteins was specifically visualized.

Full text of DOI:10.1021/cb500502n

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Article
A Rapid SNAP-tag Fluorogenic Probe Based on Environment-
Sensitive Fluorophore for No-Wash Live Cell Imaging
Tao-Kai Liu, Pei-Ying Hsieh, Yu-De Zhuang, Chi-Yang Hsia, Chi-Ling Huang,
Hsiu-Ping Lai, Hung-Sheung Lin, I-Chia Chen, Hsin-Yun Hsu, and Kui-Thong Tan
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500502n • Publication Date (Web): 08 Aug 2014
Downloaded from http://pubs.acs.org on August 12, 2014
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A Rapid SNAP-tag Fluorogenic Probe Based on Environment-
Sensitive Fluorophore for No-Wash Live Cell Imaging
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TaoꢀKai Liu,†,§ PeiꢀYing Hsieh,+,§ YuꢀDe Zhuang,† ChiꢀYang Hsia,† ChiꢀLing Huang,†
HsiuꢀPing Lai,† HungꢀSheung Lin,† IꢀChia Chen,†,‡ HsinꢀYun Hsu,*,#,+ KuiꢀThong Tan*,†,‡
†Department of Chemistry, National Tsing Hua University, 101 Sec. 2, KuangꢀFu Rd,
Hsinchu 30013, Taiwan (ROC)
‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National
Tsing Hua University, 101 Sec. 2, KuangꢀFu Rd, Hsinchu 30013, Taiwan (ROC)
#Department of Applied Chemistry and Institute of Molecular Science, National Chiaoꢀ
Tung University, No.1001 TaꢀHsueh Road, Hsinchu 30010, Taiwan (ROC)
+Center for Interdisciplinary Science (CIS), National ChiaoꢀTung University, No.1001 Taꢀ
Hsueh Road, Hsinchu 30010, Taiwan (ROC)
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Abstract
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One major limitation of labeling proteins with synthetic fluorophores is the high
fluorescence background which necessitates extensive washing steps to remove
unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on
environmentꢀsensitive fluorophore for the labeling with SNAPꢀtag proteins. The probe
exhibits dramatic fluorescence turnꢀon of 280ꢀfolds upon being labeled to SNAPꢀtag.
The major advantages of our fluorogenic probe are the dramatic fluorescence turnꢀon,
ease of synthesis, high selectivity and rapid labeling with SNAPꢀtag. Noꢀwash labeling
of both intracellular and cell surface proteins were successfully achieved in living cells
and the localization of these proteins was specifically visualized.
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Introduction
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Labeling proteins with synthetic fluorescent molecules is a powerful technique to study
protein localization, trafficking and function in live cells.1 , 2 As compared with the
fluorescent proteins, protein labeling allows for the incorporation of a wide range of
fluorophores with superior photophysical properties, coupled with the precise control of
the location and timing of labeling by the appropriate regulation of probe delivery. 3, 4
However, to achieve high signalꢀtoꢀnoise ratio for visualization and quantification,
extensive and time consuming washing of cells to remove unreacted fluorophore is
required, rendering the continuous monitoring of the dynamics of protein pools virtually
impossible.
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Fluorogenic probes, which exhibit fluorescent enhancement upon labeling to the protein
tag, are desirable in protein labeling as it helps to reduce labeling background and
substantially enhances the signalꢀtoꢀnoise ratio. However, most of the fluorogenic
probes reported to date have some limitations. For example, while the labeling of
tetracysteineꢀtag with its biarsenical probes (FIAsH/ReAsH) shows a dramatic increase
in fluorescence, live cell noꢀwash labeling is not possible due to the cytotoxicity of the
labeling reagents.5, 6 To conduct noꢀwash protein labeling in live cells, many fluorogenic
probes based on the principles of Förster resonance energy transfer (FRET) have been
reported for the labeling of proteinꢀtags such as SNAPꢀtag,7, 8, 9 BLꢀtag,10 TMPꢀtag,11 and
PYPꢀtag. 12 , 13 However, most of these probes exhibit only moderate fluorescence
enhancement upon labeling. Furthermore, FRETꢀbased fluorogenic probes normally
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require lengthy synthetic steps to prepare and have large molecular sizes which often
lead to low cell permeability and long incubation time for labeling intracellular proteins.
Thus, new fluorogenic probes which can have the combination of high selectivity, fast
labeling rate, high fluorescent turnꢀon ratio and cellꢀpermeable is highly desirable for noꢀ
wash labeling of intracellular proteins in live cells.
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Here, we described a novel fluorogenic probe BGSBD based on an environmentꢀ
sensitive fluorophore for the rapid labeling of SNAPꢀtag proteins in different cellular
compartments of living cells (Figure 1a). The SNAPꢀtag protein is one of the most
prominent labeling techniques for the covalent reaction with O6ꢀbenzylguanine (BG)
derivatives.14, 15 It is wellꢀknown for its rapid reaction rate with BG derivatives and nonꢀ
toxicity in cells. Covalent labeling of SNAPꢀtag with an affinity ligand or optical probe
has been used for protein interaction studies, 16 drug discovery, 17 superꢀresolution
imaging applications,18 and the construction of fluorescent sensors.19, 20 Most of the
SNAPꢀtag fluorogenic probes were created based on the FRET strategy by introducing
a fluorescent quencher either at the Cꢀ8 or Nꢀ9 position of guanine. However, chemical
modifications at these positions have been shown to reduce the labeling rate
dramatically and required long incubation time to label intracellular SNAPꢀtagged
proteins. Although a rapid SNAPꢀtag fluorogenic probe was reported recently by using
environmentꢀsensitive Nile red fluorophore, this strategy can only work in plasma
membrane.21 Our aim in this paper is to develop a new probe via shorter synthetic steps
in high yield which not only can achieve high fluorescence turnꢀon ratios upon SNAPꢀ
tag labeling (larger than 200ꢀfold) but can also preserve the fast labeling rate to the
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SNAPꢀtag protein. Furthermore, the probe should be cell permeable to allow for rapid
noꢀwash imaging of intracellular proteins.
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Results and Discussion
Design and synthesis of fluorogenic substrates:
In
our
strategy,
an
environmentꢀsensitive
fluorophore,
4ꢀsulfamonylꢀ7ꢀ
aminobenzoxadiazole (SBD), was attached on the benzyl amino moiety of BG to form
the fluorogenic probe BGSBD for fluorescence turnꢀon labeling of SNAPꢀtag protein.
The SBD fluorophore has been used in the applications such as protein detection and
imaging of cellular temperature. 22, 23 Although NBD is a more common environment
sensitive flurorophore with a similar benzoxadiazole chemical structure, we worked on
the SBD fluorophore as it is more sensitive to changes in polarity and the hydrogen
bonding ability of a solvent. In addition, different derivatives of BGSBD where the BG
moiety is attached either on 4‐sulfamonyl or 7‐amino position of SBD can be
synthesized for optimization (Figure 1b). Our underlying rationale for considering an
environmentꢀsensitive fluorophore to construct the fluorogenic probe for SNAPꢀtag is
that the BG binding pocket of SNAPꢀtag is hydrophobic.24, 25 In the absence of SNAPꢀ
tag, the fluorogenic probe would display weak fluorescence in aqueous buffer. Upon
covalent reaction of SNAPꢀtag with BGSBD, the environmentꢀsensitive SBD fluorophore
would be located close to the BG hydrophobic pocket to cause the SBD fluorophore to
emit stronger fluorescence.
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The synthesis toward BGSBD is rather straightforward which can be accomplished by
coupling the benzyl amino moiety of BG with the fluorophore SBD-OH (Scheme 1). The
precursor SBD-OH was prepared by first functionalization of commercially available
compound 1 with sulfonyl chloride, followed by the sequential attachment of sarcosine
and methylamine to the fluorophore. The final product BGSBD can be obtained in 5
steps with a total yield of 65 %. With this synthetic strategy, purification of any SBD-OH
intermediate was not necessary and only the final product BGSBD was purified by
reverse phase HPLC. As compared to the existing FRETꢀbased fluorogenic probes, our
approach has the advantage of having shorter synthetic steps with higher yield and
does not require any purification of the fluorophore intermediates.
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In vitro characterization of BGSBD labeled SNAP-tag protein:
BGSBD shows extremely weak fluorescence (φ = 0.0008) in aqueous PBS buffer.
However, after the SNAPꢀtag protein was labeled with the fluorophore, fluorescence
was enhanced dramatically with a high turnꢀon ratio of 280ꢀfold (φ = 0.1430, Figure 2a).
Fluorescence enhancement was so significant that it can be observed easily under a
handheld UV lamp (Figure 2a, inset). The reaction between BGSBD and SNAPꢀtag is
highly selective and produced only one single product in the SNAPꢀtag containing cell
lysate, as characterized by inꢀgel fluorescence analysis (Figure S1). Furthermore,
fluorescence enhancement occurs only in the presence of the SNAPꢀtag protein, as no
increase in fluorescence was observed in the cell lysate without SNAPꢀtag protein
(Figure S2). The hydrophobic BG binding pocket is critical as the strong fluorescence of
BGSBD labeled SNAPꢀtag was reduced dramatically in a protein denaturation buffer
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(Figure S3). As compared to the recently reported fluorescent turnꢀon labeling by using
environmentꢀsensitive fluorophores to label PYPꢀtag and TMPꢀtag where only 22ꢀfold
and 2ꢀfold enhancement was obtained, our fluorogenic probe has achieved a much
higher fluorescence turnedꢀon ratio upon labeling to the SNAPꢀtag protein.26, 27
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In bioimaging, one major concern for environmentꢀsensitive probes is the potential
background staining due to probe accumulation or nonspecific binding into lipid
membranes which can also cause fluorescence increase. To our surprise, the BGSBD
labeled SNAPꢀtag exhibits even stronger fluorescence, with higher quantum yield and
greater blueꢀshifted emission than BGSBD in hydrophobic organic solvents (Figure 2b
and Table S1). The maximum emission of BGSBD labeled SNAPꢀtag is about 37 nm
blueꢀshifted as compared to BGSBD in dioxane and shows only partial overlap with the
solvents. This feature can be of great merit for the noꢀwash labeling of BGSBD to
SNAPꢀtag in bioimaging experiments, where the background fluorescence from the
probe in the cell membrane or other hydrophobic environments can be effectively
minimized by the choice of an appropriate optical filter.
Derivatives of fluorogenic substrates for SNAP-tag protein labeling:
A series of different derivatives of BGSBD were synthesized in our attempt to optimize
the SNAP tag labeling performance (Figure 1b). The results are summarized in Table 1.
We first investigated the possibility of other environment‐sensitive fluorophores to be
used in this design for fluorescence turn‐on labeling of SNAPꢀtag protein. We attached
BG to two wellꢀknown environment‐sensitive fluorophores, NBDꢀCl and DansylꢀCl,
giving BGNBD and BGDansyl, respectively. Both BGNBD and BGDansyl showed only
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about 4ꢀfold enhancement in fluorescence upon SNAPꢀtag labeling. This result
suggests that the highly environment‐sensitive SBD fluorophore is a superior choice for
SNAP‐tag labeling to achieve significant fluorescence enhancement. It has been
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reported that guanine can act as a fluorescence quencher when it was conjugated to a
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fluorophore.
To exclude the possibility of FRETꢀbased fluorescence turn‐on
mechanism, fluorescent spectrum of SBD-OH (with no BG moiety) was measured in
PBS buffer. SBD-OH exhibits very weak fluorescence and has similar fluorescence
intensity and quantum yield as BGSBD in aqueous buffer (Table 1 and Figure S4). This
result confirms that the BG moiety is not a fluorescent quencher for SBD fluorophore
and the fluorescence enhancement of BGSBD is due to the position of SBD fluorophore
in the BG hydrophobic binding pocket.
We also synthesized derivatives of BGSBD with different linker lengths to explore the
optimal location of the SBD fluorophore in the BG hydrophobic pocket. By attaching a
C6ꢀlinker in between the SBD fluorophore and BG, BG(C6)SBD gave fluorescence
enhancement of only 5ꢀfold. The low fluorescence turn on ratio is probably due to the
longer linker chain which exposed the SBD fluorophore to the non‐hydrophobic protein
surface. Surprisingly, when the linker was shortened either by directly conjugating BG
and SBD via the sulfonyl moiety to form BG(S4)SBD or attaching BG to the 7‐amino
position of SBD to give BGSBD(N7), fluorescence enhancement was reduced from
280‐fold to merely 13ꢀ and 2ꢀfold, respectively. Therefore the proper positioning of the
SBD fluorophore in the BG binding pocket is critical and BGSBD which consists of a
sarcosine linker in between BG and the SBD fluorophore is our best fluorogenic probe,
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achieving the largest fluorescence enhancement. Finally, we studied the effect of
substituents on the 7ꢀposition of SBD fluorophore by replacing methyl amine with benzyl
amine and glycine, to give probe BGSBD(Bn) and BGSBD(Gly). Both probes gave
strong fluorescence increase upon SNAP‐tag labeling with fluorescence turn‐on ratio of
260ꢀ and 100ꢀfold, respectively. This result suggests that our fluorogenic probe design
can tolerate different substituents on 7ꢀamino position of SBD. Based on this finding,
our fluorogenic probe design not only can be applied to protein labeling, but also has
the potential to be extended to other applications, such as characterization and analysis
of ligand‐protein interactions. Currently, we are working toward developing this
fluorogenic probe design as a new analytical tool for protein detection.
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Kinetic analysis of BGSBD reaction with SNAP-tag:
A kinetic analysis of the BGSBD labeling reaction with SNAPꢀtag was carried out by
monitoring the increase of fluorescence intensity at protein and probe concentrations of
5 ꢀM each. The time required to achieve 50% labeling of protein was calculated to be
about 24 s (t1/2) and full labeling can be achieved within 3 minutes (Figure 3a). The
second order rate constant (k2) for reaction between BGSBD and SNAPꢀtag was
determined to be 7200 ± 1600 Mꢀ1sꢀ1 (Figure 3b). With this design, the probe has
successfully preserved the high labeling rate of SNAPꢀtag and the k2 value is
comparable to those native BG substrates. As compared to all the FRETꢀbased SNAPꢀ
tag fluorogenic probes reported to date, the labeling rate of BGSBD on the SNAPꢀtag is
about 10ꢀtimes faster than the fluorescent quencher attached at the Cꢀ8 position7, 9 and
around 1000ꢀtimes faster than at the Nꢀ9 position of BG.8 Furthermore, the labeling rate
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of our probe is also faster than most of the fluorogenic labeling techniques reported so
far.
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No-wash live-cell imaging of SNAP-tagged proteins with BGSBD:
To demonstrate the feasibility of applying BGSBD for noꢀwash bioimaging in different
subcellular compartments, HCTꢀ116 cells expressing SNAPꢀtag fusion proteins on the
cell surface, in cytosol and in the nucleus were investigated. We first investigated the
permeability of BGSBD to label intracellular SNAPꢀtag protein. For this goal, gene
encoding SNAPꢀtag protein only was constructed on mammalian cells expression
vector. The SNAPꢀtag expression HCTꢀ116 cells and control experiment (cells without
exogeneous SNAPꢀtag expression) were incubated with 5 ꢁM BGSBD for 30 minutes
and fluorescence images were taken without washing the cells. In the nontransfected
cells, fluorescence was hardly detected in the presence of BGSBD (Figure 4, control).
In contrast, a rapid and substantial rise in fluorescence was observed in cells
expressing SNAPꢀtag which showed that BGSBD is a membrane permeable probe to
label intracellular proteins. Toxicity test showed that the probe is nonꢀtoxic to the cells
(Figure S5). To further establish the utility of the probe for live cell imaging, we applied
the probe to label and visualize SNAPꢀtag expressed on cell surface and in nucleus. For
the labeling of cell surface proteins, a SNAPꢀtag encoding construct was prepared by
fusing SNAPꢀtag to the Nꢀterminal of transmembrane anchoring domain of plateletꢀ
derived growth factor receptor, named SNAPꢀPDGFR. For the labeling of proteins in
nucleus, gene encoding SNAPꢀtag fused with nuclear localized human histone H2B
(SNAPꢀH2B) was created. The fluorescence images of SNAPꢀPDGFR expression cells
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labeled with BGSBD revealed clear fluorescence along the plasma membrane.
Furthermore, specific labeling of SNAPꢀH2B fusion proteins in nucleus was also
observed. Coꢀstaining of the cells with Hoechst 33342 and BGSBD validated that the
probe visualized the nuclear localization of SNAPꢀH2B. Thus, these results show that
our probe can be applied to visualize a wide range of target proteins at a specific
location, either inside or outside of the cells, with no postꢀincubation washing of the
cells.
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Flow cytometric analysis of BGSBD labeled cytosolic SNAP‐tag protein:
The applications of flow cytometric techniques for the analysis of cell populations
enables scientists to evaluate cells based on both intrinsic and extrinsic properties.
Multiple characteristics of thousands of cells can be investigated within a few minutes
by using appropriate fluorescent probes such as dyes, quantum dots, or fluorescent
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proteins. 29 ,
,
However, as for liveꢀcell imaging, flow cytometric analysis of
intracellular proteins using current labeling methods also requires extensive washing to
remove the unreacted fluorophores. Due to the distinctive fluorescence emission of
BGSBD upon labeled to SNAPꢀtag and its membrane permeability, we anticipate its
potential application in flow cytometry to label intracellular proteins. To demonstrate the
feasibility of noꢀwash labeling in flow cytometric applications, 5 ꢀM BGSBD was
employed to label HCTꢀ116 cells expressing SNAPꢀtag in the cytosol for 30 minutes.
The labeled cells were then submitted to flow cytometric analysis without washout
steps. Figure 5aꢀc shows dot plots for HCTꢀ116 cells only (blank control),
nontransfected cells incubated with BGSBD (negative control) and SNAPꢀtag
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expressing cells labeled with BGSBD, respectively.
As shown in Figure 4b,
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nontransfected HCTꢀ116 cells were hardly labeled by BGSBD and displayed minimal
fluorescence background. On the other hand, robust fluorescence labeling was
observed for cells expressing cytosolic SNAPꢀtags which mostly appeared in the
fluorescenceꢀgated window (Figure 5c). The total counts of the cells that produced
fluorescence intensity above the threshold were 39, 321 and 2884 (5000 cells were
counted in each condition) for blank control, negative control and BGSBD labeled HCTꢀ
116 cell expressing cytosolic SNAPꢀtag proteins, respectively (Figure 5d).
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Conclusions
In conclusion, we have created a novel fluorogenic probe BGSBD based on the
environmentꢀsensitive fluorophore SBD for rapid labeling of SNAPꢀtag to overcome the
washout steps during protein labeling. Due to the large fluorescence enhancement and
unique emission spectra, noꢀwash labeling of intracellular proteins as well as cell
surface proteins was successfully achieved, and the localization of the proteins was
specifically visualized. This noꢀwash protein labeling technique can also be applied in
the flow cytometric analysis. The major advantages of our fluorogenic probes are the
dramatic fluorescence turnꢀon, the ease of synthesis, high selectivity, cellꢀpermeable
and rapid reaction with SNAPꢀtag. With this design, the probe BGSBD has successfully
preserved the high labeling rate of SNAPꢀtag with its BG substrates. While fluorescent
proteins will continue to be an important tool in bioimaging, the development of a noꢀ
wash chemical labeling technique may provide an alternative avenue for researchers to
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have precise control of the location and time of labeling which is crucial for realꢀtime
monitoring of high dynamic processes in living cells.
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ASSOCIATED CONTENT
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Supporting Information
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For complete experimental methods and figures see Supplementary Information. This
material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
kttan@mx.nthu.edu.tw; hyhsu99@cc.nctu.edu.tw
Author Contributions
§T.ꢀK. L. and P.ꢀY. H. contributed equally to this work.
Notes
The authors declare no competing financial interests.
Acknowledgements
We are grateful to the Ministry of Science and Technology (Grant No.: 102ꢀ2113ꢀM007ꢀ
004ꢀMY2 and 101ꢀ2113ꢀM009ꢀ006ꢀMY2) and Ministry of Education ("Aim for the Top
University Plan"; Grant No.: 102N2011E1), Taiwan (ROC) for financial support.
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(b)
BGSBD: R1 = NHMe, R2 = SarcosineꢀBG
R1
BG(C6)SBD:
R1 = NHMe, R2 = SarcosineꢀC6linkerꢀBG
N
N
BG(S4)SBD: R1 = NHMe, R2 = NHꢀBG
BGSBD(N7): R1 = NHꢀBG, R2 = N(Me)2
O
O S O
R2
BGSBD(Bn):
R1 = NHꢀBn, R2 = SarcosineꢀBG
BGSBD(Gly): R1 = GlyꢀOEt, R2 = SarcosineꢀBG
Figure 1. (a) Design of fluorescence turnꢀon probe for SNAPꢀtag protein labeling. Upon
reaction with SNAPꢀtag protein, the environmentꢀsensitive fluorophore SBD is located in
the hydrophobic BG binding pocket, whereby the surrounding hydrophobic environment
can cause the fluorophore to exhibit stronger fluorescence. In the absence of SNAPꢀtag
protein, the fluorogenic probe has low fluorescence. (b) Chemical structures of BGSBD
and its derivatives.
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Scheme 1. (a) Design of fluorogenic probe BGSBD for the fast labeling of SNAPꢀtag
protein. Upon reaction with SNAPꢀtag, the SBD fluorophore is located in the
hydrophobic BG binding pocket, whereby the surrounding hydrophobic environment can
cause the fluorophore to exhibit stronger fluorescence. (b) Reaction conditions for the
synthesis of BGSBD: (i) ClSO3H, neat, 0°C (1.5hr) then 150°C (6hr), (80%); (ii)
Sarcosine tꢀbutyl ester, TEA, DCM, RT, 10 mins, (90%); (iii) NH2Me, MeOH, RT, 1hr,
(quantitative); (iv) TFA, DCM, RT, 1hr, (quantitative); (v) BGꢀNH2, EDC.HCl, HOBt, TEA,
DMF, RT, overnight (90%).
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(b)
Figure 2. Fluorescence analyses for the labeling reactions of SNAPꢀtag with fluorogenic
probe BGSBD. (a) Fluorescence spectra of BGSBD in the absence (red line) or
presence (black line) of SNAPꢀtag protein. λex = 435 nm. The inset shows the images
of the solution in a cuvette before (left) and after labeling (right) under excitation with a
UV lamp (365 nm). The fluorescence turnꢀon ratio was calculated from the relative
fluorescence intensity of BGSBD at λem = 516 nm. Labeling condition: 5 ꢀM BGSBD
and 10 ꢀM SNAPꢀtag protein in PBS buffer (1 % DMSO) at 37°C for 30 minutes. (b)
Comparison of fluorescence response of BGSBD in various organic solvents and upon
labeled with SNAPꢀtag.
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Table 1. Spectral properties of fluorogenic probes at free and SNAPꢀtag bound states.
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free
bound
Probe
λabs
435
477
331
434
437
438
430
432
428
λem
λem
ε
Фfree
Фbound
RFI
280
4
BGSBD
584
516
13000
6300
8600
8800
8400
4800
10000
6400
4500
0.0008 0.1426
0.0230 0.0750
0.0292 0.0948
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BGNBD
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527
596
591
607
570
585
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530
530
NA
BGDansyl
SBD-OH
4
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NA
NA
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BG(C6)SBD
BG(S4)SBD
BGSBD(N7)
BGSBD(Bn)
BGSBD(Gly)
557
556
557
516
509
0.0017 0.0076
0.0013 0.0179
0.0095 0.0125
0.0050 0.3380
0.0031 0.0488
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100
(a)
NBDNHMe in ACN was used as a quantum yield reference with the value of
0.38.32 (b) The RFIs were calculated from the ratio of fluorescence intensity between the
free and SNAPꢀtag bound probe by using maximum emission wavelength of the SNAPꢀ
tag bound probe.
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(b)
Figure 3. Kinetic study of SNAPꢀtag labeling with BGSBD. (a) Time course of
fluorescence increase for the reaction of BGSBD with SNAPꢀtag. The fluorescence
intensity was recorded at 518 nm with excitation at 435 nm. (b) Fluorescence increase
of BGSBD (1 – 5 ꢀM) in labeling of SNAPꢀtag (100 nM). The inset shows the linear
relationship plot of probe concentration versus calculated kobs (R2 = 0.99).
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Figure 4. Noꢀwash liveꢀcell imaging of SNAPꢀtagged proteins labeled with BGSBD.
HCTꢀ116 cells expressing SNAPꢀtag fusion proteins at various subcellular locations. (i)
Cells without exogeneous SNAPꢀtag expression, (ii) SNAPꢀtag for both cytosol and
nucleus, (iii) SNAPꢀPDGFR for cell surface and (iv) SNAPꢀH2B for nucleus. SNAPꢀ
tagged proteins labeled with BGSBD are shown in green, nucleus labeled with Hoechst
33342 are shown in blue. The fluorescence images were obtained with excitation at 442
nm and emission at 520±20 nm.
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Figure 5. Noꢀwash flow cytometric analysis of HCTꢀ116 cells expressing cytosolic
SNAPꢀtag labeled with BGSBD. Dot plots for (a) HCTꢀ116 cells only (blank control), (b)
nontransfected cells incubated with BGSBD (negative control), (c) SNAPꢀtag
expressing cells labeled with BGSBD. Solid lines on the dot plots indicate the
fluorescence threshold. (d) The total counts of the cells that produced fluorescence
intensity above the threshold.
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SBD
BG
SBD
SNAP‐tag
SNAP‐tag
POI
POI
No‐wash Labeling
Before Labeling
After Labeling