Cy3-PEG-DBCO (MW 2000) , CAS No.C1450366

CAS: C1450366 Cat. No.: C1450366 Peso molecular: 2000 (Average)

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Descripción general

Cy3-PEG-DBCO (MW 2000) is a fluorescent probe containing Cy3 dye. Cy3-PEG-DBCO (MW 2000) contains a DBCO group that can undergo strain-promoted alkyne-azide cycloaddition (SPAAC) with molecules containing Azide groups.

Specifications

Condiciones de almacenamiento de almacenamiento

Store at -20°C

Enviado en

Ice chest + Ice pads

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Nombres e identificadores

• Peso molecular: 2000 (Average)

Application Protocols

General protocols are provided as literature guidance. Adjust to your system. Protect from light throughout.

A) Labeling an azide‑modified protein (e.g., azidohomoalanine-incorporated)

• Prepare a 10 mM stock of Cy3‑PEG‑DBCO in anhydrous DMSO.
• Exchange the protein into PBS (pH 7.4) free of sodium azide (e.g., desalting column).
• Mix to achieve 20–100 µM dye and ≤10% DMSO, using 2–5 molar eq of dye per azide site estimate.
• Incubate 1–2 h at RT with gentle mixing.
• Purify via spin desalting (7–10 kDa cutoff) or SEC. Collect the conjugate; monitor Cy3 absorbance for fraction selection.
• Characterize: UV–Vis for DOL, SDS‑PAGE with fluorescence imaging, functional assay as appropriate.

B) Live‑cell surface glycocalyx labeling after metabolic azide incorporation

• Wash cells and incubate in HBSS or PBS with 1–5 µM Cy3‑PEG‑DBCO (final DMSO ≤1%).
• React 15–30 min at 37 °C or 30–60 min at RT.
• Wash 3–4× with warm buffer; include 0.1% BSA to minimize background if needed.
• Image using TRITC/Cy3 filter set or 532–561 nm excitation.

C) Oligonucleotide or nanoparticle labeling

• Combine azide‑modified substrate in HEPES (pH 7.4) with 10–50 µM dye (≤10% DMSO).
• Incubate 1–4 h at RT; purify by SEC or dialysis suited to size.

Notes

• Always run an azide‑negative control.
• If solubility is limiting, increase DMSO incrementally up to 20% while monitoring sample stability.
• Store diluted dye solutions on ice and use within the day; for longer storage, keep concentrated aliquots at −20 °C.

Biological Roles

This product is a synthetic labeling reagent; it does not have intrinsic biological roles. The following notes describe how its components behave in biological research (literature/general):

• Cy3 fluorophore: A carbocyanine dye emitting in the orange‑red region (typical λex/λem ~550/570 nm). Used as a reporter in microscopy, flow cytometry, and blotting. Detectable with TRITC/Cy3 filter sets and many 532–561 nm laser lines.
• DBCO functional group: Chemically selective toward organic azides. It labels biomolecules that have been metabolically or chemically endowed with azide groups (e.g., azido‑sugars in glycans, unnatural amino acids in proteins, azide‑modified oligos/lipids).
• PEG linker (≈2000 Da): Enhances aqueous compatibility, reduces non‑specific hydrophobic interactions, and increases effective reach/flexibility between the dye and the biomolecule surface. PEGylation may mitigate fluorophore self‑quenching by spacing dyes (context‑dependent).

Use cases in biology (research only):

• Visualization of cell‑surface glycoconjugates after metabolic azide labeling (SPAAC on live or fixed cells).
• Tracking of azide‑functionalized antibodies, proteins, and nanoparticles for uptake/localization studies.
• Dual‑label strategies with orthogonal chemistries (e.g., combine SPAAC with NHS ester or maleimide on different sites).

Caveats:

• Sodium azide (often used as a preservative) will consume DBCO; remove it before labeling.
• Photobleaching and environmental sensitivity of cyanines require controlled illumination and antifade mounting media for imaging.
• Degree of labeling affects brightness and biological behavior; optimize to balance signal and function.

Buffer Applications

This product is not a buffering agent. However, buffer choice critically affects SPAAC labeling and fluorescence performance (literature/general guidance):

• Recommended buffers: PBS, HEPES, or Tris, pH 6.5–8.0. Ionic strength similar to physiological conditions supports protein stability.
• Avoid: Buffers or formulations containing sodium azide—it will irreversibly consume DBCO and abolish labeling. Remove by desalting/dialysis prior to reaction.
• Organic co‑solvent: 5–20% DMSO or DMF can aid solubility and reduce aggregation; titrate to minimal effective percentage.
• Protein additives: 0.01–0.05% nonionic surfactants (e.g., Tween‑20) can reduce nonspecific adsorption to plastics; verify that surfactant does not interfere with downstream assays.
• Metal ions: Unlike CuAAC, SPAAC does not require copper. Typical divalent ions (Mg2+, Ca2+) are tolerated.
• Sample cleanup: After labeling, use spin desalting columns (e.g., 7–10 kDa MWCO) or size‑exclusion chromatography to exchange into storage or assay buffer and to remove unreacted dye.

Recipe hint (example, literature):

• Prepare 1× PBS, pH 7.4. Desalt azide‑bearing protein into PBS. Add Cy3‑PEG‑DBCO from a 10 mM DMSO stock to achieve 5–50 µM final dye and ≤10% DMSO. Incubate 1–2 h at RT protected from light, then purify by SEC into PBS.

Green Alternatives

From a green chemistry perspective, Cy3‑PEG‑DBCO already enables a more sustainable workflow compared to copper‑catalyzed azide–alkyne coupling by avoiding heavy metals.

Greenness considerations (literature/general):

• Cu‑free click: Eliminates copper salts and ancillary ligands, reducing metal waste and avoiding copper‑induced biomolecule damage.
• Aqueous operation: Effective in water or water‑rich media at ambient temperatures, minimizing heating and volatile solvents.

Comparative options:

• Dyes with alternative click pairs (literature):
  • Tetrazine–TCO systems: Faster kinetics and very low working concentrations; however, tetrazines can be air‑sensitive and less stable; synthesis can be more resource‑intensive.
  • DBCO vs BCN (bicyclo[6.1.0]nonyne): BCN can be less hydrophobic and sometimes easier to handle; kinetics can differ depending on azide substrate.
• Solvent choices:
  • Prefer aqueous buffers with minimal DMSO/DMF. Where solubility allows, keep co‑solvent ≤5–10%.
  • Replace DMF with glycerol derivatives or PEG‑400 in certain prep steps if compatible (trade‑off: viscosity, purification challenges).

Small comparison table (literature/general):

• SPAAC (DBCO–azide): No metal, neutral pH, RT; k ≈ 10−2–1 M−1 s−1; dye choices broad.
• CuAAC (terminal alkyne–azide + Cu): Faster with catalyst; requires copper/ligands, scavenging, and risk of oxidative damage.
• Tetrazine–TCO: Ultra‑fast; sensitive reagents; limited buffer additives; cost and stability considerations.

Recommendations:

• Use minimal organic co‑solvent and ambient temperatures.
• Scale reactions to need; prepare concentrated DMSO stocks and dilute immediately before use to cut solvent waste.
• Dispose of dye‑containing waste responsibly; avoid light exposure to extend reagent lifetime and reduce re‑preparation.

Pharmaceutical Uses

This material is supplied strictly for research use only and is not intended for human or veterinary applications.

Formulation/manufacturing context (literature/general):

• Cy3‑PEG‑DBCO can serve as a process analytical tracer or fluorescent tag in preclinical research to follow conjugation, purification, or targeting steps of azide‑functionalized biomolecules and nanoparticles.
• PEGylated dyes are sometimes used to assess device or resin performance (e.g., SEC, HIC) during method development owing to their detectability.
• No pharmacopeial monograph or excipient status is implied. There are no approved clinical or diagnostic indications for this product.

Best practices for translational workflows (non‑clinical):

• Fully document dye‑to‑biomolecule ratios (degree of labeling), residual free dye after purification, and spectral properties in the intended buffer.
• Verify leachables/extractables from plastics and filters when scaling processes that include DMSO or DMF.
• For any GLP/GMP‑adjacent studies, qualify the lot with CoA data (identity, purity) and retain samples for stability trending.

Regulatory note: Any in vivo or clinical use requires separate-grade materials and regulatory review; this listing does not provide such authorization.

Physical Properties

Item-specific physical parameters are not provided. The following are general/literature characteristics for components to guide handling and method development. Do not treat as specifications for this item.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet. PEG–dye–DBCO conjugates are typically colored solids or viscous oils (literature).
• Solubility (general guidance):
  • High solubility in polar aprotic solvents (DMSO, DMF) (literature).
  • Usually water‑miscible upon dilution from DMSO due to PEG; final aqueous working solutions often prepared in PBS/HEPES with 5–20% DMSO or DMF to prevent aggregation (literature).
  • Limited solubility expected in nonpolar hydrocarbons; partial solubility in alcohols (EtOH, MeOH) (literature).
• Optical properties of Cy3 (typical, literature; buffer- and conjugation-dependent):
  • Absorption maximum: ~548–552 nm
  • Emission maximum: ~562–570 nm
  • Stokes shift: ~12–20 nm
  • Quantum yield: highly environment‑dependent; commonly moderate.
• LogP/charge (qualitative, literature): Amphiphilic; PEG imparts hydrophilicity; Cy3 often bears cationic centers; overall conjugate displays good aqueous compatibility.
• pKa: Not meaningful for the polymer as a whole; cationic centers on Cy3 may remain protonated across neutral pH (literature).
• Density, BP, MP, refractive index: Not specified for this item; refer to CoA/Spec Sheet.

Stability considerations (literature):

• Protect from light (photo‑bleaching of cyanines possible).
• Avoid prolonged exposure to strong acids/bases and oxidants which can degrade the dye and DBCO.
• Aqueous stability is good near neutral pH; DBCO is stable in neutral buffers but will react specifically with azides.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• What the listing indicates:
  • The product is supplied for research use only (per Research Use Note). It is not for human or veterinary use, diagnostics, or clinical procedures.
  • The name “PEG‑2000” denotes a PEG chain of nominal average molecular weight ~2000 Da; PEG materials are inherently polydisperse (literature). Consequently, the overall conjugate exhibits a distribution of masses centered around the nominal value.
• Spectral quality considerations (general):
  • Dyes for imaging are often controlled for purity of the fluorophore and low levels of non‑fluorescent byproducts. When critical, verify by HPLC and mass spectrometry and consult the CoA for acceptance criteria specific to the lot.
• Stabilizers/Counterions: Not specified for this item; refer to CoA/Spec Sheet. Cyanine dyes may be isolated as various salt forms; confirm on CoA if relevant to your assay (literature).
• Lot-to-lot consistency: For quantitative imaging/assay work, determine degree of labeling (DOL) and verify absorbance spectra for each lot to ensure consistent performance.

Documentation to request when qualifying lots:

• CoA including purity profile (HPLC), identity (HRMS), and water/solvent content if relevant.
• SDS for safety classification and handling.
• If needed, spectral scan of the lot (absorption/emission) under your planned buffer conditions.

Reaction and Applications

This reagent is designed for copper‑free click bioconjugation via strain‑promoted azide–alkyne cycloaddition (SPAAC) between DBCO and organic azides.

Key applications (literature/general):

• Bioconjugation of azide‑modified biomolecules: proteins (via azidohomoalanine, azide‑bearing linkers), glycoconjugates (metabolic azide labeling), nucleic acids (azide handles), lipids, and polymers.
• Live‑cell and in‑situ labeling: SPAAC proceeds in aqueous media at neutral pH and ambient temperature without Cu catalysts, minimizing cytotoxicity relative to CuAAC.
• Imaging/analytics: Cy3 provides bright orange‑red fluorescence compatible with TRITC/Cy3 channels in microscopy, flow cytometry, Westerns, and microarrays.

Mechanistic/kinetic notes (literature):

• SPAAC forms a 1,2,3‑triazole through a concerted [3+2] cycloaddition. DBCO exhibits second‑order rate constants typically in the 10−2–1 M−1 s−1 range with simple azides, depending on medium and substitution.
• Reaction is highly selective for azides over common biological functional groups; thiols, amines, and carboxylates are tolerated.

Practical guidance:

• Use 1.2–5.0 molar equivalents of Cy3‑PEG‑DBCO relative to azide sites to drive completion; optimize for your target to limit over‑labeling.
• Conduct at pH 6–8, room temperature; typical conversions within 30 min to several hours. For sterically hindered targets, extend to overnight at 4–25 °C.
• Avoid sodium azide in buffers; remove by desalting before labeling.
• Purify conjugates by spin desalting, SEC, or dialysis to remove free dye. Verify coupling by absorbance ratio (Cy3 peak vs protein at 280 nm), MS (if applicable), or gel fluorescence.

Controls:

• Include an azide‑negative control to assess non‑specific adsorption and autofluorescence.
• Perform small‑scale trials to optimize equivalents and solvent content.

Reaction Conditions

Typical SPAAC labeling conditions for DBCO–azide coupling are provided as literature/general guidance. Optimize for your system.

• Solvent/buffer: PBS, HEPES, or Tris, pH 6.5–8.0; avoid sodium azide. Include 5–10% DMSO (up to 20% if needed) to maintain solubility.
• Concentrations:
  • Biomolecule (e.g., protein): 0.5–10 mg/mL depending on stability.
  • Dye (Cy3‑PEG‑DBCO): 5–200 µM typical; start with 2–5 molar eq relative to azide sites.
• Temperature: 20–25 °C (RT). For sensitive samples, 4 °C with extended times.
• Time: 0.5–4 h for many systems; sterically hindered substrates may require overnight.
• Kinetics: Second‑order; apparent rate increases with higher concentrations and better mixing. DBCO typically shows k ≈ 10−2–1 M−1 s−1 vs simple azides (literature), slower for crowded protein sites.
• Light: Protect from light to preserve fluorophore integrity.
• Workup: Remove excess dye by spin desalting (7–10 kDa cutoff), SEC, or dialysis. Confirm removal by monitoring Cy3 absorbance in flow‑through.
• Analytics:
  • UV–Vis: Measure Amax near ~550 nm to quantify dye; correct for overlap at 280 nm when calculating DOL for proteins (use literature correction factors or empirically determine).
  • SDS‑PAGE with in‑gel fluorescence or fluorescence SEC to verify conjugation.
  • MS/peptide mapping for site identification when applicable.

Compatibility notes:

• Tolerates salts and common additives; avoid primary azide scavengers (sodium azide) and high concentrations of competing strained alkynes.
• For live‑cell labeling, keep DMSO ≤1–2% v/v and wash thoroughly post‑labeling.

Safety and Handling

Hazard classification for this specific item is not provided; always consult the product SDS for authoritative guidance.

• GHS/CLP: Not specified for this item; refer to SDS. In absence of classification, handle as a laboratory chemical of unknown hazards.
• Primary hazards (general):
  • Cyanine dyes may cause irritation to skin/eyes; avoid inhalation/ingestion (literature).
  • DBCO is a strained alkyne; not explosive as supplied, but can be reactive toward azides—store away from sodium azide or azide‑containing preservatives.
• PPE: Lab coat, safety glasses, and nitrile gloves. Use in a fume hood when weighing or making DMSO/DMF stocks to avoid inhalation of aerosols/solvent vapors.
• Handling tips:
  • Work under low light or amber lighting; store solutions in amber vials.
  • Use anhydrous solvents for stock solutions to minimize hydrolytic degradation; keep containers tightly closed to limit moisture uptake by PEG.
• Incompatibilities (general): Strong oxidizers, strong acids/bases, and reactive azides in bulk. Avoid buffers containing sodium azide during storage or prior to labeling, as it will quench DBCO reactivity.
• First aid overview (general):
  • Skin/eye contact: Rinse with water for at least 15 minutes; remove contaminated clothing; seek medical attention if irritation persists.
  • Inhalation: Move to fresh air; seek medical advice if symptoms occur.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
• Waste: Collect organic dye wastes and DMSO/DMF rinses as hazardous organic waste per institutional and local regulations.

Solvent Selection

Cy3‑PEG‑DBCO is amphiphilic: the PEG chain promotes water compatibility, while the dye/DBCO segments prefer polar aprotic media. Choose solvents to balance solubility, stability, and biocompatibility.

• Primary stock solvent (recommended, literature): Anhydrous DMSO or DMF at 5–20 mM. These solvents solubilize the dye and DBCO efficiently and allow subsequent dilution into aqueous buffers.
• Aqueous working solutions:
  • Buffers: PBS, HEPES, or Tris (pH ~6.5–8). For SPAAC, buffer composition is flexible; avoid sodium azide.
  • Co‑solvent: 5–20% DMSO/DMF can suppress aggregation and surface adsorption; titrate to the lowest amount compatible with your biology.
• Alcohols: MeOH/EtOH can dissolve the dye but are less favored for biomolecule conjugations due to protein denaturation at higher percentages.
• Nonpolar solvents: Generally poor choices; limited solubility and risk of dye aggregation.
• pH effects (literature): Cyanine spectral properties can shift slightly with solvent polarity and pH; maintain consistent conditions for quantitative imaging.

Small comparison (general):

• DMSO: Excellent solubility, biocompatible in small %; hygroscopic.
• DMF: Similar to DMSO; more volatile odor and potential reactivity with some plastics.
• Aqueous buffer alone: May be feasible at low micromolar levels; risk of aggregation/adsorption without a co‑solvent.

Practical tips:

• Warm gently to room temperature and vortex/sonicate briefly to dissolve; avoid heating above ~40 °C to minimize dye degradation (literature).
• Filter (0.22 µm PTFE) if particles persist; test for dye loss to filters before critical experiments.

Storage and Reconstitution

• Storage conditions (as provided): Store at −20 °C. Shipments are made in an ice chest with ice pads. Protect from light.
• Form supplied / appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Reconstitution (general guidance):
  • Dissolve in anhydrous DMSO or DMF to prepare concentrated stocks (e.g., 5–20 mM). Vortex and, if needed, brief sonication to aid dissolution. Avoid heating above ~40 °C (literature).
  • Filter through 0.22 µm PTFE if particulates persist; confirm minimal dye loss.
  • For aqueous working solutions, dilute stocks into buffer immediately before use; avoid freeze‑thaw of dilute solutions.
• Aliquoting: Upon receipt, equilibrate to room temperature before opening to prevent condensation. Prepare small, single‑use aliquots under low light to minimize freeze‑thaw and moisture uptake by PEG.
• Light/moisture protection: Store in amber vials with desiccant where possible. Minimize headspace to limit oxidation and water ingress.
• Shelf life: Not specified for this item; refer to CoA/Spec Sheet. As a general practice, monitor absorbance spectra for degradation (peak broadening/shift) over time.
• Thawing/handling: Thaw frozen stocks on ice or at RT protected from light. Mix gently before use.
• Compatibility: Avoid storage in buffers containing sodium azide; it will irreversibly consume the DBCO functionality.

For any stability‑indicating requirements, qualify storage and use intervals empirically under your laboratory conditions.

Structure and Identity

Cy3-PEG-DBCO (MW 2000) is a fluorescent cyclooctyne probe: a Cy3 cyanine fluorophore linked through a polyethylene glycol chain to a dibenzocyclooctyne (DBCO) group for copper‑free click chemistry (SPAAC).

• SKU: C1450366
• Product name: Cy3-PEG-DBCO (MW 2000)
• CAS: C1450366 (catalog reference)
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: The name indicates a PEG segment of nominal 2000 Da; exact average MW of the full conjugate is not specified for this item; refer to CoA/Spec Sheet.
• SMILES / InChIKey: Not specified for this item; refer to CoA/Spec Sheet.

Structural features (general description):

• Fluorophore: Cy3 (carbocyanine, orange‑red emitting). Typically contains two indoleninium/quinolinium-type heteroaromatic rings bridged by a polymethine chain (literature).
• Linker: PEG(≈2000 Da) providing hydrophilicity, flexibility, and aqueous solubility; polydisperse by nature (literature).
• Reactive handle: DBCO (dibenzocyclooctyne), a strained cyclooctyne fused to two benzene rings, enabling rapid, catalyst‑free [3+2] cycloaddition with organic azides (SPAAC) (literature).
• Overall architecture: Cy3–(PEG2000)n–DBCO, typically an amphiphilic macromolecule; no defined stereocenters in PEG; dye/DBCO may possess multiple aromatic rings (literature).

2D structure in words (literature/general): cationic Cy3 chromophore at one end, a flexible ethylene oxide repeat chain (~45 EO units typical for PEG2000) in the middle, and a hydrophobic, strained dibenzocyclooctyne ring at the terminus.

Synthetic Utility

While primarily a bioconjugation reagent, Cy3‑PEG‑DBCO also functions as a chemoselective handle in synthetic/analytical workflows (literature/general):

• Functional group reactivity: The DBCO strained alkyne undergoes SPAAC with organic azides to form 1,2,3‑triazoles without catalysts. Highly tolerant of alcohols, amines, thiols, carboxylates, and many protecting groups.
• Orthogonality: Can be combined with NHS ester, maleimide, thiol–ene, or oxime chemistries in multistep labeling to introduce multiple modalities (e.g., fluorophore + affinity tag) on a single scaffold.
• Polymer/materials modification: Efficiently labels azide‑bearing polymers, surfaces, and nanoparticles for imaging or tracking, leveraging PEG to enhance dispersibility.
• Analytical tagging: Installation of a strong chromophore/fluorophore aids in HPLC/CE detection and MS confirmation (with appropriate methods), enabling kinetic studies of azide content or mapping of modification sites after proteolysis.
• Spacer effects: The PEG2000 linker reduces steric hindrance and can improve access to buried azide sites relative to short linkers, often increasing effective conversion.

Limitations/cautions:

• DBCO can undergo side reactions with highly electron‑rich dipoles at elevated temperatures or in neat organic media; maintain mild, aqueous conditions when possible.
• Excess hydrophobic dye domains may promote aggregation on some biomolecules; the PEG spacer mitigates but does not eliminate this—optimize equivalents and solvent composition.

Target Specificity

This product is not a biological affinity reagent (e.g., antibody) and has no antigen/epitope specificity.

Chemical specificity (literature/general):

• Reactive partner: Organic azides (–N3). The DBCO group reacts rapidly and selectively with azides via SPAAC to form a stable triazole linkage.
• Selectivity profile: Minimal cross‑reactivity with common biological nucleophiles (amines, thiols, alcohols, carboxylates) under neutral aqueous conditions. Sodium azide acts as a competitive sink and must be excluded.

No clone, isotype, species reactivity, or epitope information applies to this item.