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| Targets |
Cy3-N3 does not bind to a biological target; it is a chemical labeling reagent. The azide group (-N3) is a bioorthogonal functional group that reacts specifically with alkynes via click chemistry. The Cy3 fluorophore provides orange fluorescence (Ex/Em ~550/570 nm) for detection. This approach allows labeling of biomolecules without interfering with their native functions.
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| ln Vitro |
In vitro, Cy3-N3 is used for fluorescent labeling of biomolecules (peptides, proteins, oligonucleotides, carbohydrates) through click chemistry. For CuAAc, the alkyne-bearing molecule is incubated with Cy3-N3 (1-5 equivalents) in the presence of CuSO4 (1 mM) and sodium ascorbate (2-5 mM) in PBS buffer (pH 7.4) with 10-20% DMSO for 1-12 hours at 37degC. For SPAAC, Cy3-N3 is mixed with DBCO- or BCN-conjugated molecules in PBS (pH 7.4) for 2-4 hours at 37degC. The labeled products are purified by dialysis or HPLC and used in downstream assays.
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| ln Vivo |
CY3-N3 is not administered directly for in vivo activity. It is used to label targeting ligands through click chemistry. The Cy3-labeled conjugates (e.g., antibodies, peptides) can be administered to animal models for fluorescence imaging in the visible range (Ex/Em ~550/570 nm). However, Cy3 is less suitable for deep-tissue imaging compared to NIR dyes (e.g., Cy5, Cy7) due to higher tissue absorption and autofluorescence at shorter wavelengths.
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| Enzyme Assay |
For in vitro click chemistry conjugation, Cy3-N3 is used in enzyme-free or CuAAc reactions. Standard protocol for CuAAc: Dissolve 1 micromol of alkyne-functionalized biomolecule in 200 microL of degassed PBS (pH 7.4, 0.2 M). Add 1-1.5 micromol Cy3-N3 (in 10-20 microL DMSO). Prepare fresh 100 mM sodium ascorbate and 10 mM CuSO4 solutions. Add 20 microL of 100 mM sodium ascorbate (final 10 mM) and 20 microL of 10 mM CuSO4 (final 1 mM) to the reaction mixture (final volume 200-250 microL). Incubate at 37degC for 4-12 hours with gentle shaking under an inert atmosphere (N2 or Argon). For SPAAC, dissolve 1 micromol DBCO- or BCN-conjugated molecule in 200 microL PBS (pH 7.4). Add 1-1.2 micromol Cy3-N3 in 10-20 microL DMSO. Incubate at 37degC for 2-4 hours. Monitor conjugation by TLC (silica gel, 10% methanol in dichloromethane) or by LC-MS. Purify by size-exclusion chromatography (e.g., Sephadex G-25) or dialysis (3.5-10 kDa cutoff). Determine labeling efficiency by measuring absorbance at 550 nm (Cy3) and 280 nm (protein).
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| Cell Assay |
For cell-based assays, Cy3-labeled probes are used for live-cell or fixed-cell imaging. Cells (e.g., 5×10⁴ cells/well in 8-well chamber slides) are seeded and cultured overnight. Add Cy3-labeled antibody or peptide (1-10 microg/mL) to the culture medium and incubate for 1-4 hours at 37degC. Wash cells with PBS (3×). For live-cell imaging, add fresh medium and image immediately using a fluorescence microscope with Cy3 filter (Ex 540-560 nm, Em 570-590 nm). For fixed-cell imaging, fix with 4% paraformaldehyde for 15 minutes, wash, counterstain nuclei with Hoechst 33342 or DAPI, mount, and image. For antibody binding and internalization studies, consider using confocal microscopy for z-stack acquisition.
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| Animal Protocol |
For in vivo animal studies, Cy3-labeled conjugates are typically administered via intravenous or intratumoral injection to mice. However, due to the visible-range fluorescence of Cy3 (Ex/Em ~550/570 nm), applications are often limited to subcutaneous or surface tumors where tissue penetration is less critical. Typical dose: 50-100 microg of Cy3-labeled antibody per mouse in 100-200 microL PBS. In vivo fluorescence imaging is performed using an imaging system with excitation at 540-560 nm and emission at 570-590 nm. Biodistribution can be assessed by ex vivo organ imaging after euthanasia. For deep-tissue imaging, NIR dyes (Cy5, Cy7) are generally preferred over Cy3.
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| ADME/Pharmacokinetics |
Cy3-N3 has a molecular weight of 712.88 Da. The compound is supplied as a solid and should be stored at -20degC, protected from light and moisture. Powder is stable for up to 3 years at -20degC and 2 years at 4degC. Stock solutions in DMSO (10-20 mM) should be stored at -80degC for up to 6 months or at -20degC for up to 1 month, and should be protected from light to prevent photobleaching. Cy3-N3 is soluble in DMSO (e.g., 10-50 mg/mL) and moderately soluble in water (with sulfonated versions; the non-sulfonated version requires organic solvents). For in vivo formulations, common vehicles include 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, or DMSO:cornoil (1:9).
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| Toxicity/Toxicokinetics |
Cy3-N3 is considered non-toxic at typical research concentrations (microM to low mM range for conjugation reactions). Standard laboratory safety practices should be followed: use of gloves, lab coat, and eye protection; avoid inhalation of dust. This product is for research use only and not for human diagnostic or therapeutic applications. No significant toxicity has been reported. For in vivo studies, standard safety monitoring (body weight, behavior) should be performed.
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| References | |
| Additional Infomation |
Cy3-N3 (Cyanine3 azide) is a fluorescent labeling reagent for click chemistry. Key features: (1) Cy3 fluorophore with excitation/emission maxima at approximately 550/570 nm, providing bright orange-red fluorescence; (2) Azide group for bioorthogonal conjugation via CuAAc or SPAAC; (3) Sulfonated versions provide excellent water solubility. Common applications include labeling of oligonucleotides (e.g., for FISH), peptides, proteins (e.g., for immunofluorescence), and small molecules. The compound is also used in metabolic labeling studies (e.g., glycan labeling via azide-bearing sugars). Purity is typically ≥98% by HPLC. CAS number: 1621101-43-6.
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| Molecular Formula |
C34H44N6O7S2
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| Molecular Weight |
712.88
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| Appearance |
Brown to black 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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4028 mL | 7.0138 mL | 14.0276 mL | |
| 5 mM | 0.2806 mL | 1.4028 mL | 2.8055 mL | |
| 10 mM | 0.1403 mL | 0.7014 mL | 1.4028 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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