| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
| Targets |
Cyanine5 NHS ester is a fluorescent labeling agent; it does not have a specific biological receptor target. The NHS ester group is chemically reactive toward the ε-amino group of lysine residues and the N-terminal alpha-amine of proteins/peptides. Once covalently conjugated to a biomolecule, the cyanine 5 fluorophore (excitation approx. 649 nm, emission approx. 670 nm) can be excited by a red laser or LED source to emit far-red fluorescence. The dye is used for visualizing and tracking the distribution of labeled molecules in vitro and in vivo.
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| ln Vitro |
End-capping Poly(β-amino esters) (pBAEs) backbones are achieved by reacting the free amino group on the cysteine amino acid with cyanine-5 NHS ester amine-reactive red emitting fluorescent dye[1].
The in vitro activity of the dye is measured by its ability to label biomolecules efficiently without causing functional impairment. After conjugation to a protein (e.g., IgG antibody, BSA, transferrin), the degree of labeling (DOL, moles of dye per mole of protein) is calculated by measuring absorbance at 280 nm (protein) and 649 nm (dye). Typically, a DOL of 2-10 is considered optimal. The labeled conjugate retains the biological activity of the original protein, which is verified by a functional assay (e.g., antigen binding ELISA for labeled antibodies). No direct pharmacological activity exists. |
| ln Vivo |
Cyanine5 NHS ester-labeled probes are used extensively for in vivo imaging studies in small animals (mice, rats). After intravenous injection of a Cy5-labeled antibody or peptide, the probe's biodistribution and tumor-targeting ability can be monitored non-invasively using an in vivo imaging system (IVIS) with appropriate excitation/emission filters (Ex/Em ~ 640/680 nm). The far-red wavelength minimizes tissue autofluorescence and allows penetration depths of 5-10 mm, enabling imaging of superficial tumors and organs. The dye has low toxicity at typical imaging doses (0.1-1 nmol per mouse).
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| Enzyme Assay |
The NHS ester chemistry is characterized in simple chemical model reactions. The reactivity of Cyanine5 NHS ester iodide with a model primary amine (e.g., n-butylamine, 10 mM) in PBS (pH 7.2-8.5) is monitored by HPLC or LC-MS. The reaction proceeds rapidly at room temperature (completion within 30-60 minutes). The stability of the NHS ester in aqueous solution (hydrolysis rate) is determined by incubating the dye in PBS (pH 7.4) at 37degC and measuring the disappearance of the NHS ester peak (retention time shift) over 0-8 hours by HPLC. The half-life for hydrolysis at pH 7.4 is typically 30-90 minutes.
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| Cell Assay |
To label a protein with Cyanine5 NHS ester: 1) Dissolve the protein (e.g., IgG, 1-10 mg/mL) in 0.1 M sodium bicarbonate buffer (pH 8.5). 2) Dissolve Cyanine5 NHS ester iodide in anhydrous DMSO (or DMF) to a concentration of 10 mM. 3) Add 5-20 molar equivalents of the dye solution to the protein solution (for a final DMSO concentration of ≤5%). 4) Incubate the mixture at room temperature for 1-2 hours with gentle stirring or mixing, protected from light. 5) Remove unreacted dye by size exclusion chromatography (e.g., Sephadex G-25 column, Zeba desalting columns) equilibrated with PBS, or by dialysis (3 changes of PBS over 24 hours). 6) Collect the labeled protein eluate. 7) Determine the DOL by measuring absorbance at 280 nm (protein) and 649 nm (dye) and using appropriate correction factors. The labeled protein is stored at 4degC with 0.02% sodium azide as preservative, protected from light.
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| Animal Protocol |
Cy5 NHS ester is not used for animal dosing. However, the Cy5-labeled probe derived from it is used for in vivo imaging. A typical protocol: Female athymic nude mice bearing subcutaneous xenograft tumors (e.g., 4T1 breast cancer, 150 mm3) are injected intravenously via tail vein with 100-200 microL of a Cy5-labeled tumor-targeting antibody (e.g., anti-EGFR, anti-HER2) in PBS at a dose of 1-5 mg protein/kg (approx. 10-50 microg dye per mouse). At predetermined time points (e.g., 1, 4, 8, 12, 24, 48 hours post-injection), mice are anesthetized with isoflurane (1.5-2% in O2). Whole-body fluorescence images are acquired using an in vivo imaging system (Ex 640 nm, Em 680 nm, exposure time 0.1-1 s). After final imaging (usually 48-72 hours), mice are euthanized, and major organs (tumor, liver, kidney, spleen, heart, lung, brain) are excised and imaged ex vivo to confirm biodistribution. Tumor-to-background ratios are calculated.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of the dye itself are not evaluated because the NHS ester reacts immediately upon injection with serum proteins. The resulting covalent protein-dye conjugate inherits the PK properties of the carrier protein. For antibody-dye conjugates, the circulation half-life in mice is typical of the antibody (e.g., 3-7 days for IgG) but can be slightly reduced due to the hydrophobic dye. For small peptide-dye conjugates, the half-life is minutes to a few hours, depending on peptide size. The dye is metabolically stable and excreted primarily via the liver/biliary and renal pathways depending on the conjugate size.
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| Toxicity/Toxicokinetics |
Cyanine5 NHS ester iodide is a fluorescent labeling reagent. The acute toxicity of the free dye (unconjugated) is low. Cytotoxicity assays on mammalian cells (e.g., HeLa, HEK-293) show no significant reduction in viability at concentrations up to 10 microM (IC50 > 50 microM). At labeling doses used for in vivo imaging (nanomole quantities per mouse), no adverse effects such as weight loss, hunched posture, or changes in motor activity are observed. No skin or eye irritation is reported for the dye. The iodide counterion does not contribute additional toxicity. However, the NHS ester is an alkylating agent in vitro, but it is rapidly hydrolyzed or reacted in vivo. As with any reactive chemical, avoid inhalation and skin contact; use PPE.
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| References | |
| Additional Infomation |
Cyanine5 NHS ester iodide is a red-fluorescent labeling reagent belonging to the cyanine dye family. The NHS ester group reacts specifically with primary amines to form stable amide bonds. Cyanine5 is preferred over fluorescein (FITC) or rhodamine because it exhibits less photobleaching, higher brightness, and minimal autofluorescence from biological samples in the far-red/near-infrared region. The iodide salt form is typically more water-soluble than the chloride form. The product is for research use only (not for human diagnostics or therapy). Store as a dry powder at -20degC, protected from light and moisture. The stock solution should be prepared fresh or stored in small aliquots to avoid repeated freeze-thaw cycles, which can accelerate hydrolysis.
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| Molecular Formula |
C36H42IN3O4
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| Molecular Weight |
707.64
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| Related CAS # |
Cyanine5 NHS ester chloride;1032678-42-4;Cyanine5 NHS ester bromide;1653991-59-3
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| Appearance |
Yellow to brown solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :~110 mg/mL (~155.45 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.75 mg/mL (3.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.75 mg/mL (3.89 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4131 mL | 7.0657 mL | 14.1315 mL | |
| 5 mM | 0.2826 mL | 1.4131 mL | 2.8263 mL | |
| 10 mM | 0.1413 mL | 0.7066 mL | 1.4131 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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