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Cell-Based Applications of Living Colors® Proteins
Valuable tools in multicolor screening applications
• Easy detection compatible
with diverse instrument platforms
including the BD™ Pathway HT
confocal bioimaging system
A
Untreated
B Treated (bottom of cell)
C Treated (middle of cell)
• Ideally suited for subcellular
protein trafficking and highcontent screening assays
• No need for added cofactors
or substrates
Cell-based assays have undergone tremendous development in recent years
as current screening technologies evolve
toward cost-effectiveness and biological
significance. This shift in focus strongly
emphasizes more relevant, informative,
and complex cell-based systems over the
traditional in vitro-based assays.
Fluorescent proteins, historically wellestablished tools for noninvasive and
real-time studies in live cells, are now
being used in a variety of reagent-free
homogenous assays that address current
screening needs. The spectral properties of
fluorescent proteins make them extremely
well suited for multicolor applications in
fluorometry, flow cytometry, and fluorescence microscopy—fluorescent proteins
have great potential in both optimizing and accelerating drug discovery and
screening processes.
We offer a panel of Clontech Novel
Fluorescent Proteins (NFPs) suited
for precisely these applications. They
consist of the Reef Coral Fluorescent
Proteins (RCFPs)—AmCyan1, ZsGreen1,
ZsYellow1, DsRed, AsRed2, and
HcRed1, as well as our new monomeric
DsRed-Monomer (red) and AcGFP1
(green) proteins (1–3). DsRed-Monomer
is the only commercially available red
monomeric fluorescent protein suitable
for dual color fusion applications
with AcGFP1.
Figure 1. GPCR activation monitored by Living Colors DsRed2 fusion proteins in the
Transfluor assay. U-2 OS cells were cotransfected with the ß2-adrenergic receptor and a
ß-arrestin-2-DsRed2 fusion protein and treated for 30 min with 1 µM isoproterenol. Cells
were analyzed by confocal microscopy at 63X magnification. Panel A. Untreated cells.
Panels B and C. Images of stimulated cells were taken at either the interface of the cell
and the plate, or at a cross section through the cell.
Our comprehensive collection of distinct,
novel fluorescent proteins covers an extensive emission spectrum that ranges from
489 nm (AmCyan1) to 618 nm (HcRed1)
(4, 5). No cofactors or substrates need to
be added for detection, which further simplifies and streamlines your experiments.
Wide range of applications
Our fluorescent proteins are powerful
tools for establishing a wide range of assays (reviewed in 4). RCFPs and AcGFP1
have been used successfully as fusion
partners when fused to a targeting signal,
and are ideal markers for cells and organelles (6–7). Monomeric AcGFP1 and
DsRed-Monomer are the best candidates
for studies requiring full-length protein
fusions—they perform extremely well in
fusions with full-length actin, tubulin,
and insulin (data not shown).
Furthermore, RCFPs have been successfully expressed in a diverse range—plant
to animal—of whole body organisms (1;
data not shown). RCFPs that are bright
and/or available in a destabilized format,
such as ZsGreen1, HcRed1, and DsRedExpress, are also perfect transcriptional
reporters (4, 6). The RCFPs, in particular
ZsGreen, have also been shown to be
powerful and sensitive biosensors for
monitoring live-cell proteasome activity
as well as markers for in vitro migration
assays modeling metastatic tumor invasion
(8; data not shown).
Clontech Laboratories, Inc. • www.clontech.com
CR682048
Ideal for high-content
screening assays
The examples highlighted here complement the previously published uses of
Clontech NFPs as specialized screening
tools. NFPs can be used to establish reliable, high-content cell-based assays such
as monitoring G-protein coupled receptor
(GPCR) activation. Use of a DsRed2 fusion to the ß-arrestin-2 regulatory protein
provides an elegant Norak Transfluor™
assay for monitoring ß2-adrenergic receptor activation (Figure 1). In the absence
of agonist, the DsRed2-ß-arrestin-2 fusion protein is distinctly localized to the
cytoplasm. Upon agonist stimulation, the
fusion moves to the plasma membrane
where it participates in receptor desensitization and clathrin-coated pit internalization. The formation of coated pits
indicates activation of the ß2-adrenergic
receptor.
Reprinted from Clontechniques January 2005 Cell-Based Applications…continued
A
B
Figure 2. Four color visualization of Living Colors Novel Fluorescent Proteins using the
BD™ Pathway Confocal Bioimager. Panel A. HEK 293 clonal cell lines stably expressing
AmCyan1, ZsGreen1, ZsYellow1, or HcRed1 were mixed and plated into the same well and
then imaged at 20X magnification using the BD™ Pathway Bioimager. Chroma Technology filter sets were used to separate the signal of each individual fluorescent protein
(HQ440/30, 470DCXR, and HQ488/35 for AmCyan1; HQ488/10, 84100 beam splitter, and
HQ540/50 for ZsGreen1; HQ500/40, 530DCLP, and HQ555/40 for ZsYellow1; and HQ575/50x,
Q610LP, and HQ640/50m for HcRed1). Panel B. HEK 293 cells were transiently transfected
with constructs encoding AmCyan1-Peroxi (peroxisome), ZsYellow1-Nuc (nucleus), and
HcRed1-Mito (mitochondria) fusion proteins. Slides were imaged at 60X magnification
with an oil objective using the method described above.
Multiple NFPs can identify
distinct cell subpopulations
and intracellular structures
Notice to Purchaser
For all licensing information, visit www.clontech.com
Multicolor detection within a mixed
population of cells expressing AmCyan1,
ZsGreen1, ZsYellow1 or HcRed1 can be
performed using a confocal microscopy
platform such as the BD™ Pathway HT
Confocal Bioimager (Figure 2, Panel A).
Our NFPs can also be used to label and
easily distinguish subcellular organelles
and compartments from one another in
live cells (Figure 2, Panel B).
Clontech Fluorescent Proteins, with their
multiplexing capabilities and uniquely
suited qualities, should play an ever
increasing role in the development of
even more sophisticated cell-based drugdiscovery and screening processes.
Living Colors Products
Size
Cat. No.
pAcGFP1-N1 Vector
20 µg
632469
pAcGFP1-C1 Vector
20 µg
632470
pAmCyan1-N1 Vector
20 µg
632442
pAmCyan1-C1 Vector
20 µg
632441
pAsRed2-N1 Vector
20 µg
632449
pAsRed2-C1 Vector
20 µg
632450
pDsRed2-N1 Vector
20 µg
632406
pDsRed2-C1 Vector
20 µg
632407
pDsRed-Express-N1 Vector
20 µg
632429
pDsRed-Express-C1 Vector
20 µg
632430
pDsRed-Monomer-N1 Vector
20 µg
632465
pDsRed-Monomer-C1 Vector
20 µg
632466
pHcRed1-N1/1 Vector
20 µg
632424
pHcRed1-C1 Vector
20 µg
632415
pZsGreen1-N1 Vector
20 µg
632448
pZsGreen1-C1 Vector
20 µg
632447
pZsYellow1-N1 Vector
20 µg
632445
pZsYellow1-C1 Vector
20 µg
632444
References
1. Matz, M. V., et al. (1999) Nature Biotechnol.
17:969–973. Erratum in: Nature Biotechnol.
(1999) 17:1227.
2. Gurskaya, N. G., et al. (2001) FEBS Lett.
507:16–20.
3. Gurskaya, N. G., et al. (2003) Biochem. J.
373:403–408.
4. Haugwitz M., et al. (2003) Genet. Eng. News
23(21).
5. Verkhusha, V. V. & Lukyanov, K. A. (2004)
Nature Biotechnol. 22(3):289–296.
6. Reef Coral Fluorescent Protein Vectors
(July 2003) Clontechniques XVIII(3):67.
7. BD Living Colors™ HcRed1 Localization Vectors
(July 2003) Clontechniques XVIII(3):8.
8. BD Living Colors™ Cell Lines (April 2004)
Clontechniques XIX(2):2–4.
Clontech Laboratories, Inc. • www.clontech.com
Living Colors® Related Products
• Anti-RCFP Polyclonal Pan Antibody
(Cat. No. 632475)
• Full-Length ZsGreen Polyclonal Antibody
(Cat. No. 632474)
• DsRed Monoclonal Antibody
(Cat. Nos. 632392 & 632393)
• DsRed Polyclonal Antibody
(Cat. No. 632397)
• HcRed Polyclonal Antibody
(Cat. No. 632452)
• BD™ Pathway Bioimager (inquire)
For Research Use Only. Not for use in diagnostic
or therapeutic procedures. Not for resale. Clontech,
Clontech logo, and all other trademarks are the property
of Clontech Laboratories, Inc. Clontech is a Takara Bio
Company. Copyright 2006.
Reprinted from Clontechniques January 2005