Cy5 mono reactive dye

Manufactured by GE Healthcare

Sourced in Japan, United Kingdom, United States

Cy5 Mono-Reactive Dye is a fluorescent dye used in various laboratory applications. It is designed to be covalently attached to biomolecules, enabling their detection and analysis. The dye has an excitation maximum at 649 nm and an emission maximum at 670 nm, making it suitable for use in fluorescence-based techniques.

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Lab products found in correlation

Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Nanodrop 1000, by Thermo Fisher Scientific (2 mentions) Human oligo chip 25k, by Toray (1 mentions) Amino allyl arna kit, by Thermo Fisher Scientific (1 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions) Cy3 mono reactive dye, by GE Healthcare (1 mentions) Imject alum, by Thermo Fisher Scientific (1 mentions) C57bl 6 mice, by Jackson ImmunoResearch (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Amicon ultra centrifugal filter unit, by Merck Group (1 mentions) Vero cell, by American Type Culture Collection (1 mentions) Mircury lna microrna array power labeling kit, by Qiagen (1 mentions) Goat f ab 2 anti mouse igκ, by Southern Biotech (1 mentions) Goat f ab 2 anti humanfc5μ, by Jackson ImmunoResearch (1 mentions) Alexafluor 647 nhs esters, by Thermo Fisher Scientific (1 mentions) Alexa fluor 405, by Thermo Fisher Scientific (1 mentions) Linear pei 25 kda, by Polysciences (1 mentions)

6 protocols using cy5 mono reactive dye

miRNA Profiling Using Microarray

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RNAs (including miRNAs) were extracted using the mirVana™ miRNA Isolation Kit (Life Technologies Japan Ltd.) according to the recommendations of the manufacturer. Microarray analysis was carried out using the Human Oligo chip 25 K (ID QH0ZG35) by Toray Co. (http://www.3d-gene.com/en/about/; Tokyo, Japan) as described previously [9 (link)]. In brief, total RNA was amplified using an Amino Allyl aRNA kit (Ambion). RNA from cells was labeled with Cy3 Mono-Reactive Dye (GE Healthcare Japan Co., Tokyo, Japan), and control RNA was labeled with Cy5 Mono-Reactive Dye (GE Healthcare). After purification, each 1-μg sample was mixed and hybridized at 37 °C for 16 h. After washing, the hybridized chip was scanned using a 3D-Gene Scanner 3000 (Toray Co.). Background was subtracted from the raw data and the values were normalized according to a median Cy3/Cy5 ratio of 1.

Ueki S., Murakami Y., Yamada S., Kimura M., Saito Y, & Saito H. (2016). microRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line. BMC Cancer, 16, 732.

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Fluorescent Labeling of Immune Proteins

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Cy3 labeling of biotinylated moth cytochrome c (MCC) peptide (residues 88–103), ovalbumin (OVA) (Sigma, St. Louis, MO), BSA (Sigma), CGG (EMD Millipore, Billerica, MA), and streptavidin (SAv) was carried out with Cy3 maleimide and amine-reactive labeling kits (GE Healthcare, Little Chalfont, UK). NP (4-hydroxy 3-nitrophenylacetyl)-phycoerythrin (PE) was prepared using NP-O succinymidyl ester (NP-OSu) (Biosearch Technologies, Petaluma, CA). NP-chicken gamma globulin (NP-CGG) and NP-bovine serum albumin (NP-BSA) (Biosearch Technologies) were fluorescently labeled with Cyanine 5 (Cy5) on amine groups (Cy5 Mono-Reactive Dye, GE Healthcare).
C57BL/6 mice were purchased from Jackson Laboratories and housed in the Stanford Animal Facility for at least one week before use. IL-17f^{Thy1.1/Thy1.1} mice and G8/Rag2^−/− TCR transgenic mice were bred and housed in the pathogen-free Stanford Animal Facility. All experiments were performed in accordance with the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee. 200 μg each of Cy3-CGG and CGG in aluminum hydroxide (Imject Alum; Thermo Scientific, Waltham, MA) per mouse and subcutaneous immunization were used in all studies.

Zeng X., Meyer C., Huang J., Newell E.W., Kidd B.A., Wei Y.L, & Chien Y.H. (2014). Gamma delta T cells recognize haptens and mount a hapten-specific response. eLife, 3, e03609.

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Rotavirus Particle Purification and Labeling

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RVs (table S3) were grown in Vero cells (American Type Culture Collection) in the presence of trypsin (39 (link)). RV TLPs were purified from MA104 cell lysates as described (40 (link)). DLPs were prepared by treating TLPs with 5 mM EDTA for 20 min at 37°C. VP2–enhanced green fluorescent protein (eGFP)/VP6 particles were prepared as described (41 (link)). TLPs (CDC-9) were labeled with Cy5 as described (42 (link)) with modifications. TLPs (100 μg) were washed with 10 mM Hepes (pH 8.2), 5 mM CaCl₂, and 140 mM NaCl, labeled at 4:1 molar ratio of Cy5 monoreactive dye (GE Healthcare) to TLPs at room temperature for 1 hour followed by the addition of tris-HCl (pH 8.8) to a final concentration of 50 mM. Labeled viruses were purified by dialysis using Amicon Ultra Centrifugal filter unit (Millipore). The integrity of TLP-Cy5 was determined by electron microscopy (43 ).

Nair N., Feng N., Blum L.K., Sanyal M., Ding S., Jiang B., Sen A., Morton J.M., He X.S., Robinson W.H, & Greenberg H.B. (2017). VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans. Science translational medicine, 9(395), eaam5434.

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Comprehensive mRNA and miRNA Expression Profiling

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mRNAs were labeled with Cy5 mono-reactive dye (GE Healthcare, Little Chalfont, UK) and purified according to the manufacturer's protocol (3DGENE mRNA CyDye label v2, Toray Industries, Tokyo, Japan). The concentration of labeled RNAs was determined by a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) before hybridization onto microarray chips (Human Oligo Chip 25k ver. 2.10., Toray Industries). Hybridization and subsequent washing were performed according to the manufacturer's protocol (3DGENE mRNA hybridization v2, Toray Industries). For miRNA analysis, miRNA was labeled, hybridized, and washed according to the manufacturer's protocol (3DGENE miRNA label Hybridization 4plex v1, Toray Industries) using the miRCURY LNA microRNA Array Power Labeling kit (Exiqon, Vedbaek, Denmark) and Human miRNA oligo chip (Human miRNA Chip ver. 16.1.0.0, Toray Industries). The intensity of labeled mRNAs or miRNAs was analyzed with the 3D-Gene Scanner 3000 system with auto gain, auto focus, and auto analysis settings.

Ito M., Sun S., Fukuhara T., Suzuki R., Tamai M., Yamauchi T., Nakashima K., Tagawa Y.I., Okazaki S., Matsuura Y., Wakita T, & Suzuki T. (2017). Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation. Oncotarget, 8(33), 53899-53915.

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Fluorescent Labeling of Protein Probes

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Model antigens used in solution or on PMS were goat anti‐mouse Igκ F(ab')₂ (Southern Biotech) for mouse splenic B cells and goat F(ab')₂ anti‐human Fc5μ (Jackson Immunoresearch) for Ramos and DG75 cells. The antibodies were biotinylated using EZ‐Link NHS‐LC‐LC‐biotin (Pierce) and conjugated to one of several fluorophores ‐ Cy5 Monoreactive dye (GE Healthcare), AlexaFluor 405 or AlexaFluor 647 NHS esters (Thermo Fisher) in Sodium Carbonate buffer, according to the manufacturer's instructions. Excess dye was removed using Zeba 7K MWCO desalting columns (Pierce, Thermo Fisher). Mouse transferrin (Rockland) was also fluorescently labeled and biotinylated for TfR internalization assays.

Malinova D., Wasim L., Newman R., Martínez‐Riaño A., Engels N, & Tolar P. (2021). Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses. EMBO Reports, 22(9), e51328.

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Cy5 Labeling of Linear 25 kDa PEI

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Linear 25 kDa PEI (25 kDa, Polysciences, Warrington, PA, USA) was covalently labeled with Cy5 monoreactive dye (Amersham, GE Healthcare, Little Chalfont, UK) by suspending one vial with 2 mg of PEI dissolved in 1 mL PBS at pH 8.0, adapted from [16] (link). Reaction was performed for 4 h at RT under continuous mixing. To eliminate Cy5 excess, the resultant Cy5-labeled PEI solution was dialyzed through a 5 kDa-cutoff cellulose membrane (Sigma Aldrich, San Luis, MO, USA) in a 2 L PBS solution and pH 7.4 at RT for 7 d. Dialysis solutions were changed every 24 h. PEI labeling was confirmed by absorbance measurement with Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Maintenance of PEI activity was confirmed by its consistency with the standard transient transfection protocol. Cy5-labeled PEI was stored at -20 °C protected from light.

González-Domínguez I., Grimaldi N., Cervera L., Ventosa N, & Gòdia F. (2019). Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells. New biotechnology, 49.

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