Cyanine3 (cy3)

Manufactured by Lumiprobe

Sourced in United States, Germany

Cy3 is a fluorescent dye commonly used in molecular biology applications. It has an excitation wavelength of 550 nm and an emission wavelength of 570 nm, making it suitable for a variety of labeling and detection methods.

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20 protocols using cyanine3 (cy3)

Immobilization of OCP on Cy3/Cy5-loaded SBA-15

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Experiments were carried out using 4 μM stock solutions of cyanine dyes Cy3 and Cy5 (Lumiprobe) in Milli-Q water (pH 7 for Cy3 and pH 6.5 for Cy5). In order to obtain a 1:9 Cy3:Cy5 ratio, 0.1 mL of Cy3 stock solution was added to 0.9 mL of Cy5 stock solution. This 1:9 ratio was selected to maximise the detectable emission from Cy5 [25 (link)]. The overall 1.0 mL mixed solution was put in contact with 10 mg of SBA-15-80-NH2 powder and gently stirred for 60 min. After centrifugation, Cy3/Cy5-loaded SBA-15-80-NH₂ nanoparticles were collected and analysed with UV-Vis and fluorescence spectroscopy.
To carry out the subsequent immobilisation of OCP on Cy3/Cy5-loaded SBA-15-80-NH₂ nanoparticles, 1 mL of OCP(CAN) 0.2 mg/mL was added to the dye-loaded SBA-15-80-NH₂ pellet and gently stirred for 4 h on a moving plate, in the dark, to avoid the photo-activation of the protein. Then, the sample was centrifuged for 30 min at 8000 rpm, and the OCP/Cy3/Cy5 SBA-15-80-NH₂ analysed with UV-Vis and fluorescence spectroscopy.

Leccese S., Calcinoni A., Wilson A., Kirilovsky D., Carbonera D., Onfroy T., Jolivalt C, & Mezzetti A. (2023). Orange Carotenoid Protein in Mesoporous Silica: A New System towards the Development of Colorimetric and Fluorescent Sensors for pH and Temperature. Micromachines, 14(10), 1871.

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SARS-CoV-2 N protein phase separation

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The dimeric N constructs with removed cellular nucleic acids were labeled by fluorescence dye Oregon-Green488 (Invitrogen, 2,161,802) with a molar ratio 10:1 between protein and fluorescence dye in labeling buffer 50 mM NaPhosphate pH 7.0, 50 mM NaCl. The mixtures were incubated at 25 ℃ for 1 h. To remove the excess fluorescence dye, the resulting mixture was buffer-exchanged with labeling buffer using concentrator columns with 10 kDa cutoff (Ultrafiltration Centrifugal Tube, Millipore). The SARS-CoV-2 5' UTR and 3' UTR were labeled by fluorescence dye Cy3 (Lumiprobe, #41,070) following the Cy3 labelling protocol [79 (link)]. The labeled proteins and RNAs were quantified using a NanoDrop One^C (Thermo Scientific).
Oregon-Green488 labeled N constructs were diluted to a final concentration of 25 μM in phase separation buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl) containing 10% PEG 3350, and RNAs were added with a protein to RNA molar ratio of 100:1. Then the mixture was incubated at room temperature for 5 min. A total of 10 μL solution was transferred onto the glass slide, and images were collected using a Zeiss-Axio Observer 7 microscope.

Wang Y., Ling X., Zhang C., Zou J., Luo B., Luo Y., Jia X., Jia G., Zhang M., Hu J., Liu T., Wang Y., Lu K., Li D., Ma J., Liu C, & Su Z. (2023). Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly. Molecular Biomedicine, 4, 16.

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Investigating Pancreatic Cancer Metastasis and Drug Delivery

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All animal work was performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee. For the in vivo orthotopic xenograft assay, AsPC-1 cells (9 × 10⁵ cells/mouse) that stably expressed luciferase were directly injected into the pancreas of female BALB/c nude mice (6 weeks old; Nara Biotech, Seoul, Korea). The luciferase activity in the mice was immediately imaged after injection to confirm that the cancer cells were successfully implanted. S-Benp was orally administered 5 days per week (50 mg/kg) from day 1 (the day after cancer cell injection). Tumor growth was monitored under a live animal imaging system (PHOTONE IMAGER, Biospace, Nesles-la-Vallée, FRANCE) after injecting luciferin (15 mg/mL, Gold Bio, St. Louis, MO, USA). On day 21, the mice were sacrificed and evaluated for metastatic tumor growth in major organs, such as the pancreas, liver, spleen, and kidney.
For drug delivery investigation, AsPC-1 cells (9 × 10⁶ cells/mouse) were subcutaneously injected into the right flanks of nude mice (6-week-old female BALB/c mice). A single dose of Cy3 (Lumiprobe) or Cy3-S-Benp was intravenously administered at 5 mg/kg when the average tumor volume reached 267.2 ± 18.1 mm³. Tumors were collected at 3 and 6 h after treatment for immunohistochemistry and the cellular thermal shift assay.

Jang H.J., Yoon Y.J., Choi J., Lee Y.J., Lee S., Cho W., Byun W.G., Park S.B., Han D.C, & Kwon B.M. (2022). S-Benproperine, an Active Stereoisomer of Benproperine, Suppresses Cancer Migration and Tumor Metastasis by Targeting ARPC2. Pharmaceuticals, 15(12), 1462.

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Fluorescent Dyes for Biomolecular Labeling

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Cyanine3 (NHS ester), Cyanine3 (maleimide), Cyanine5 (NHS ester) and Cyanine5 (maleimide) were purchased from Lumiprobe. Puromycin was purchased from Cayman Chemical. UltraPure™ 1M Tris–HCI pH 7.5 was obtained from Invitrogen. Other common materials and reagents were purchased from Sigma or Amresco.

Wu B., Zhang H., Sun R., Peng S., Cooperman B.S., Goldman Y.E, & Chen C. (2018). Translocation kinetics and structural dynamics of ribosomes are modulated by the conformational plasticity of downstream pseudoknots. Nucleic Acids Research, 46(18), 9736-9748.

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Synthesis of Fluorescent GLP-1 Receptor Ligands

Cited 2 times

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Exendin4(9–39)-S39C was generated
as previously reported using solid phase peptide synthesis.^{19 (link),29 (link)} TSTU activation of CPY-6-COOH and reaction with 1-(2-amino-ethyl)-pyrrole-2,5-dione
(TFA salt, Aldrich) yielded Mal-CPY. Maleimide-conjugated CF488A (Aldrich),
Cy3, and Cy7 (both Lumiprobe) were purchased from commercial vendors.
Coupling to peptides was performed using thiol-maleimide chemistry
in PBS, before characterization of novel compounds using HRMS, and
purity (>95%) measurement using HPLC. Because fluorophores may
exhibit
environmental dependence upon receptor binding, for which extinction
coefficients and quantum yields are challenging to determine, we instead
highlight manufacturer measures for CF488-Mal, Cy3-Mal, CPY-6-COOH,
and Cy7-Mal. In any case, all probes performed similarly when bound
to SNAP-GLP1R or endogenous receptor, both in cells and tissues. Details
for synthesis including characterization of LUXendin492, LUXendin551, LUXendin615, and LUXendin762 are provided in the Supporting Information.

Ast J., Novak A.N., Podewin T., Fine N.H., Jones B., Tomas A., Birke R., Roßmann K., Mathes B., Eichhorst J., Lehmann M., Linnemann A.K., Hodson D.J, & Broichhagen J. (2022). Expanded LUXendin Color Palette for GLP1R Detection and Visualization In Vitro and In Vivo. JACS Au, 2(4), 1007-1017.

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Tracking TRIM25 and G3BP1 Phase Separation

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TRIM25 and G3BP1 proteins were labeled by fluorescence dye Oregon-Green488 (Invitrogen, 2161802) and Cy3 (Lumiprobe, #41070), respectively. Purified proteins and fluorescence dyes were incubated with a molar ratio of 10:1. After reaction at 25 °C for 1 h, the excess fluorescence dye was removed by desalting column. The labeled proteins were concentrated to 10 mg/ml and stored in −80 °C.
The phase separation assays were performed at the room temperature, following an established protocol^{78 (link)}. MBP-tagged TRIM25 (WT and ∆PTFG) and G3BP (WT, GDM and TBM) were mixed with 20 ng nucleic acids (dsRNA, ssRNA, dsDNA or ssDNA) in solution containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 8% v/v Ficoll. TEV protease was added at 0 min to start the reaction. The formation of droplets was observed using Olympus FV3000 confocal microscope with a 60x objective.

Shang Z., Zhang S., Wang J., Zhou L., Zhang X., Billadeau D.D., Yang P., Zhang L., Zhou F., Bai P, & Jia D. (2024). TRIM25 predominately associates with anti-viral stress granules. Nature Communications, 15, 4127.

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Fluorescent Oligonucleotide Synthesis and Purification

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Tetrahydrofuran (THF)-containing oligonucleotides were synthesized with biotin and amino modifications, and purified by HPLC (Integrated DNA Technologies, USA). A thymine glycol (Tg) containing oligonucleotides were synthesized with biotin and amino modifications (Bioneer, South Korea), and subsequently purified with PAGE. The oligonucleotides were fluorescently labeled at the amine groups by standard assays using Cy3 or Cy5 NHS esters (Lumiprobe, USA) for smFRET measurements (Supplementary Table S1).

Yi G., Sung Y., Kim C., Ra J.S., Hirakawa H., Kato T.A., Fujimori A., Kim H, & Takata K.I. (2023). DNA polymerase θ-mediated repair of high LET radiation-induced complex DNA double-strand breaks. Nucleic Acids Research, 51(5), 2257-2269.

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Fluorescent Labeling of Duplex DNA

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DNA oligonucleotides were custom synthesized with biotin and amino modifications, and purified by HPLC (Integrated DNA Technologies, USA). The oligonucleotides were fluorescently labeled at the amine groups by standard assays using Cy3 or Cy5 NHS esters (Lumiprobe, USA), and unreacted dyes were removed by ethanol precipitation. To generate duplex DNA molecules, single-stranded DNAs were mixed in a 1:1 ratio, annealed at 95 °C for 1 min, and then slowly cooled to room temperature for 1 h.

Sohn B.K., Basu U., Lee S.W., Cho H., Shen J., Deshpande A., Johnson L.C., Das K., Patel S.S, & Kim H. (2020). The dynamic landscape of transcription initiation in yeast mitochondria. Nature Communications, 11, 4281.

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Purification and Labeling of Muscle Tropomyosin

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Rabbit skeletal and chicken gizzard smooth muscle tropomyosin was isolated and purified according to previously published procedures (Jancso and Graceffa 1991 (link), Graceffa 1999 (link), Graceffa 2000 (link)). Purified skeletal muscle tropomyosin consists of either two α-α chains (Tpm1.1st (a.b.b.a)) or one α and one β (Tpm2.2st (a.b.b.a)) chain, while smooth muscle tropomyosin contains mostly heterodimers with one α (Tpm1.4sm (a.a.b.d)) and one β (Tpm2.1sm/cy (a.b.a.d)) chain (Geeves, Hitchcock-DeGregori et al. 2014 ). Non-muscle tropomyosin isoform Tpm1.6cy (a.b.b.d) (Geeves, Hitchcock-DeGregori et al. 2014 ), common name Tm2, was generously provided by Dr. Alan Huang. All three isoforms were dissolved in Phosphate Buffered Saline and reduced by reacting with 5 mM DTT for 3 hours followed by dialysis against 3–4 changes of buffer. Tissue purified tropomyosin was labeled at Cys-190 with a small molecule fluorescent probe, Alexa-532, which has little or no effect on actin binding (Bacchiocchi and Lehrer 2002 (link), Bacchiocchi, Graceffa et al. 2004 (link)). Labeling on cysteine-190 was achieved by incubation of tropomyosin with a 5-fold molar excess of the maleimide derivative of AlexaFluor-532 (Invitrogen) or Cy3 (Lumiprobe) overnight at 4°C. The labeled proteins were purified by gel filtration chromatography using Sephadex G25 (Supplemental Figure S4).

Schmidt W.M., Lehman W, & Moore J.R. (2015). Direct Observation of Tropomyosin Binding to Actin Filaments. Cytoskeleton (Hoboken, N.J.), 72(6), 292-303.

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Labeling and Tracking Myxobacterial OM Proteins

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We developed a method to label myxobacterial OM proteins with Cy3 (Lumiprobe) to test for transfer between cells. Live cells were grown to mid-log phase, washed, and incubated with Cy3 dyes in appropriate liquid medium as follows: for M. xanthus, 50 µg/ml Cy3 and 1-h incubation in CTT; for S. cellulosum, 1 µg/ml Cy3 and 15-min incubation in medium M. After incubation, the stained cells were washed three to five times in fresh medium and resuspended to ∼5 × 10⁸ cells/ml. Recipient cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Invitrogen) as described previously (5 (link)). Cy3-labeled donors and CFDA-SE-labeled recipients were then mixed (1:1 ratio) and incubated on agarose pads consisting of 1.2% agarose in the appropriate medium. Cell mixtures were incubated in the dark at 33°C for 0.5 h for M. xanthus cells and for ≥4 h for S. cellulosum cells before imaging was performed. To test for OME in C. crocatus Cm c5, a lipid dye transfer assay was done as described previously (9 (link)). Donor cells were labeled with a red fluorescent DiD lipid dye (lipophilic tracer sampler kit; Invitrogen), whereas the recipients were labeled with CFDA-SE (Invitrogen).

Cao P., Wei X., Awal R.P., Müller R, & Wall D. (2019). A Highly Polymorphic Receptor Governs Many Distinct Self-Recognition Types within the Myxococcales Order. mBio, 10(1), e02751-18.

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