Cy5 PEG NHS - MW 2000 Da

Cat. No.: C163508

GRADE & PURITY MW 2000 Da

Storage

A l'abri de la lumière,A conserver à -20°C

Shipped In

Glacière + blocs de glace

Vue d’ensemble

Le polyéthylène glycol marqué au Cy5 est un dérivé du PEG fluorescent dans le proche infrarouge qui présente une absorption maximale à 650 nm et une émission autour de 670 nm. Le dérivé PEG marqué au Cy5 peut être facilement repéré grâce à sa couleur bleue et à sa forte fluorescence. Une fonctionnalité supplémentaire permet à ces dérivés PEG fluorescents d'être facilement conjugués à d'autres molécules ou surfaces matérielles.

Specifications

Spécifications et pureté

MW 2000 Da

Conditions de stockage de stockage

A l'abri de la lumière,A conserver à -20°C

Expédié en

Glacière + blocs de glace

Propriétés chimiques et physiques

• Solubilité: Blue or dark blue solid or viscous liquid depends on molecule weight;Soluble in regular aqeous solution as well as most organic solvents
• Sensibilité: light sensitive

Application Protocols

Tested applications and validated protocols: Not specified for this item; refer to CoA/Spec Sheet and SDS.

Guidance: For general reaction setup and handling recommendations, see the Reaction & Applications and Reaction Conditions sections in this page. Always validate protocols on a small scale with your specific substrate and instrumentation.

Biological Roles

This product is a synthetic amine-reactive fluorescent labeling reagent, not a natural metabolite.

General information about the Cy5 fluorophore in research (literature/general):

• Function: Cy5 is a far-red cyanine dye with excitation/emission typically around 645–650/660–670 nm. Far-red emission reduces cellular autofluorescence and photodamage relative to shorter wavelengths, improving signal-to-noise in imaging and flow cytometry.
• PEG spacer role: The PEG linker increases aqueous compatibility, reduces hydrophobic interactions with biomolecular surfaces, and can mitigate dye–dye self-quenching when multiple labels are installed.
• Bioconjugation target: NHS ester chemistry labels primary amines on proteins (lysines, N-termini), peptides, amine-modified oligos, and surfaces. The resulting amide is chemically robust under physiological conditions.
• Applications: In vitro fluorescence assays, single-molecule studies, FRET pairs (Cy3–Cy5), super-resolution methods that accept cyanine probes, and surface-based biosensors.

Important notes:

• No biological activity or specificity is implied—labeling depends on amine accessibility and reaction conditions.
• Photophysics can depend strongly on local environment (polarity, proximity to aromatic residues, oxygen level). Validate brightness and photostability in the intended assay matrix.
• Research Use Only: This reagent is not intended for diagnostic or therapeutic use in humans or animals.

Buffer Applications

Applicability: Relevant for setting up bioconjugation reactions using the NHS ester functionality.

General buffer guidance (literature/best practice):

• Recommended conjugation buffers (amine-free):
  • 0.1 M Sodium carbonate/bicarbonate, pH 8.3–8.5 (enhances amine nucleophilicity while balancing NHS hydrolysis).
  • 50 mM Sodium phosphate, pH 7.8–8.0 with 100–150 mM NaCl (gentler on sensitive proteins; slightly slower kinetics).
  • 50 mM HEPES, pH 7.8–8.2 (amine-free zwitterionic buffer alternative).
• Avoid: Tris, glycine, ammonium salts, and other primary/secondary amines which compete with the intended amine targets and consume NHS ester. Also avoid azide only if it may interfere with downstream assays; azide does not react with NHS esters but may affect some enzymes.
• Organic cosolvent: Keep DMSO/DMF under 10–20% v/v to preserve biomolecule structure; pre-dissolve dye in anhydrous solvent before buffer addition.
• pH control: Monitor pH before and after adding dye stock; DMSO/DMF have minimal buffering but concentrated acidic/basic stocks can shift pH. Maintain temperature at 20–25°C for predictable kinetics.

Recipes (illustrative):

• 0.1 M Carbonate buffer pH 8.3: Mix 0.1 M Na2CO3 and 0.1 M NaHCO3 to target pH; filter 0.22 µm.
• 50 mM Phosphate buffer pH 7.8: Prepare from Na2HPO4/NaH2PO4; add 150 mM NaCl if needed for ionic strength.

Note: These are general-purpose buffers; this item’s specific buffer compatibility is not specified—confirm with small-scale trials.

Green Alternatives

Perspective: Bioconjugation with NHS esters commonly uses polar aprotic solvents (DMSO/DMF) and mild aqueous buffers. Greener choices aim to minimize hazardous solvents and waste while maintaining performance.

Options and trade-offs (literature/general):

• Use minimal organic cosolvent: Prepare concentrated DMSO stock and add the smallest volume necessary to reach target dye concentration in aqueous buffer (<10% v/v is often sufficient). This reduces VOCs and exposure.
• Prefer DMSO over DMF where feasible: DMSO is generally regarded as having a more favorable environmental, health, and safety profile than DMF or NMP. Viscosity may limit microfluidic applications.
• Aqueous-only activation is impractical: NHS esters hydrolyze in water; purely aqueous formulations drastically reduce shelf life and coupling efficiency.
• Buffer selection: Carbonate or borate buffers at pH 8–8.5 avoid volatile amines and enable efficient coupling without added catalysts.
• Sulfonated/longer-PEG variants: If solubility in water is limiting, choose dye variants with higher inherent hydrophilicity (e.g., sulfo-Cy5 NHS or longer PEG). This reduces reliance on organic cosolvent during labeling.

Comparison snapshot (general):

• DMF: Excellent solvent power; reproductive toxicity concerns; more stringent exposure controls.
• DMSO: Broad solvency, relatively benign; can enhance dermal absorption—use gloves and avoid skin contact.
• NMP: Good solvency but more hazardous; typically avoid when DMSO/DMF suffice.

Waste minimization:

• Scale reactions precisely, use microspin desalting, and quench residual NHS ester before disposal to reduce reactive waste streams.

Pharmaceutical Uses

Item-specific pharmacopeial status and approved uses: Not specified for this item; refer to CoA/Spec Sheet.

General context (literature/industry practice):

• Role: Cy5 PEG NHS is an activated labeling reagent used to prepare fluorescently tagged biomolecules for in vitro analytical applications (e.g., assay development, QC, mechanistic studies). It is not an excipient and is not intended for clinical administration.
• Manufacturing/analytical workflows: May be used to generate reference standards, tracer molecules for ligand-binding assays, or fluorescent probes for process development analytics. Resulting labeled biomolecules can support in vitro diagnostics research, subject to regulatory validation when applicable.
• Quality considerations: For any use in regulated workflows, traceable documentation (CoA, stability studies, impurity profile) and method validation (identity/purity of conjugates, residual free dye) are required. Photostability and batch-to-batch consistency of spectral properties should be established.
• Formulation of conjugates: Labeled proteins are often stored in buffered saline with stabilizers (e.g., sugars, glycerol) and light protection. The dye reagent itself is kept dry, cold, and protected from light until use.

Disclaimer: No therapeutic or clinical claims are made. This product is provided strictly for research use only.

Physical Properties

Item-specific physical data:

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• Other specifications (mp, bp, density, solubility limits, refractive index, water/peroxide/metal content): Not specified for this item; refer to CoA/Spec Sheet.

Typical properties for Cy5-PEG-NHS reagents (literature/general, not product specifications):

• State: Commonly supplied as a dark green to blue-purple solid or lyophilized powder; color can vary with counterion and concentration.
• Solubility: Readily dissolves in anhydrous DMSO or DMF to form concentrated stocks (e.g., 5–20 mM). After dilution into aqueous buffers (pH 7–9), solubility is improved by the PEG spacer; however, hydrolysis of the NHS ester proceeds in water.
• Spectral characteristics for Cy5 chromophore: Abs λmax typically ~645–650 nm; Em λmax ~660–670 nm in aqueous media; ε often ~2.5–2.7 × 10^5 M−1 cm−1 (values depend on exact structure and medium).
• Stability: NHS ester half-life in aqueous buffer is pH- and temperature-dependent (literature: tens of minutes at pH ~8–8.5, 20–25°C). Hydrolysis yields the non-reactive carboxylate.
• Partitioning: PEGylation increases hydrophilicity relative to non-PEG cyanines; nonetheless, the dye core remains hydrophobic, which can drive aggregation at high concentrations without surfactant or protein present.

Caveat: Use spectral or purity data from the CoA for this specific lot when quantifying concentration or degree of labeling.

Quality and Grades

Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.

General context for dye-activated esters (literature/industry practice):

• High-purity requirements: For quantitative bioconjugation and analytical applications (e.g., flow cytometry, fluorescence quantification), materials are typically provided at high chemical purity (often ≥95% by HPLC) with narrow impurity profiles to minimize background fluorescence and non-specific labeling. Exact values for this SKU are not provided here.
• Stabilizers: Some NHS ester dyes may be packaged with desiccants and under inert gas to limit hydrolysis; liquid stabilizers are uncommon because they can interfere with conjugation. No stabilizer information is provided for this item.
• Documentation: The Certificate of Analysis typically includes identity confirmation (HPLC/LC–MS), purity, residual solvents, and spectral data (λmax, ε). Use the lot-specific CoA as the authoritative specification for this product.
• Light and temperature controls: This SKU’s storage conditions specify protection from light and storage at −20°C, consistent with preserving NHS ester integrity and cyanine photostability.

Recommendations:

• Verify purity by analytical HPLC before sensitive assays if required.
• For quantitative labeling, determine concentration spectrophotometrically using the dye’s extinction coefficient (consult the lot CoA for the exact ε used in calculations).

Reaction and Applications

Scope (literature/general for Cy5 PEG NHS):

• Primary application: Covalent labeling of primary amines on biomolecules (lysine side chains, N-termini of proteins/peptides, amine-functionalized oligonucleotides or surfaces) via NHS ester chemistry to form stable amide bonds.
• Use cases: Fluorescence imaging, in-gel fluorescence, blot detection, microarray spotting, flow cytometry, FRET studies, and surface immobilization where a PEG spacer reduces dye–biomolecule interactions and self-quenching.

Reaction overview:

• Mechanism: Nucleophilic acyl substitution of the NHS ester by a deprotonated amine, expelling N-hydroxysuccinimide.
• pH window: Optimal 7.5–8.5 in aqueous buffers; below pH ~7 reactivity is reduced, above pH ~9 hydrolysis accelerates and can cause protein damage.
• Competing reaction: Hydrolysis by water to the carboxylate (non-reactive). Work quickly with freshly prepared solutions.

Practical tips:

• Exclude competing nucleophiles (avoid Tris, glycine, ammonium salts). Prefer carbonate (0.1 M, pH 8.3) or phosphate buffers (50 mM, pH 7.8–8.0) with 50–150 mM NaCl.
• Degree of labeling (DOL): For proteins, start with 2–10 molar equivalents of dye per monomer. Incubate 30–60 min at room temperature protected from light. Quench residual NHS ester with ethanolamine (after conjugation and before purification) if desired.
• Purification: Remove free dye by desalting columns (e.g., Sephadex G-25), spin filters (10–30 kDa MWCO), or HPLC for small peptides.
• Analytics: Determine DOL by UV–Vis using the dye’s ε and protein’s A280 correction factor (obtain from CoA/literature for the exact variant). Record spectra to monitor hydrolysis (appearance of NHS peak loss) and conjugation.

Reaction Conditions

Item-specific conditions: Not specified for this item; refer to CoA/Spec Sheet.

General conditions for NHS-ester labeling with Cy5 PEG NHS (literature/best practice):

• Stock solution: 5–20 mM in anhydrous DMSO or DMF; prepare fresh immediately before use.
• Buffer: 50 mM phosphate (pH 7.8–8.0) or 0.1 M carbonate/bicarbonate (pH 8.3–8.5), amine-free, 20–25°C. Maintain ionic strength (100–150 mM NaCl) for protein stability.
• Stoichiometry: 2–10 molar equivalents of dye per protein monomer (adjust to target DOL). For small-molecule amines, 1.1–1.5 equiv amine with 1.0 equiv NHS ester in anhydrous solvent, with 2–3 equiv DIPEA or similar base.
• Time: 30–60 min for proteins at RT with gentle mixing; 1–4 h for sterically hindered substrates. Monitor for hydrolysis by UV–Vis or LC.
• Quench (optional): Add 10–50 mM ethanolamine (pH 8.0) after conjugation to consume residual NHS ester before purification.
• Purification: Desalting columns (G-25) or ultrafiltration (10–30 kDa MWCO) for proteins; prep HPLC for peptides/small molecules.
• Light control: Wrap vessels in foil or use amberware to prevent photobleaching.

Performance notes:

• NHS hydrolysis half-life decreases with increasing pH and temperature; minimize delays after adding dye to buffer.
• Avoid organic fractions >20% v/v during protein labeling to preserve native structure; validate for each target.

These are general guidelines. Optimize conditions empirically for your substrate and consult the lot-specific documents for any provided parameters.

Safety and Handling

Item-specific hazard data:

• GHS Classification: Not specified for this item; refer to SDS.
• Signal Word / Pictograms / H-Statements: Not specified for this item; refer to SDS.

General safety guidance for NHS-activated cyanine dyes (literature/best practice):

• Handle in a fume hood or well-ventilated area. Avoid inhalation of dust and contact with skin/eyes.
• PPE: Laboratory coat, nitrile gloves (change frequently when handling in DMSO/DMF), and safety glasses or face shield for weighing. Use amber glassware or foil wrap to protect from light.
• Incompatibilities: Moisture, nucleophiles (amines, alcohols), and basic aqueous solutions promote rapid hydrolysis of the NHS ester. Avoid buffers containing primary/secondary amines (e.g., Tris, glycine). Avoid prolonged exposure to elevated temperature and light (photo-bleaching of cyanines).
• First aid (overview; consult SDS): In case of skin contact, wash with soap/water. Eye contact: rinse cautiously with water for several minutes and seek medical attention. If inhaled, move to fresh air. If swallowed, rinse mouth and seek advice.
• Spill/cleanup: Avoid dust formation; collect solid with disposable towels or damp wipes. For solutions, absorb with inert material; dispose of as hazardous chemical waste per institutional procedures.
• Waste: Collect dye-containing organic solutions (DMSO/DMF) in halogen-free organic waste streams unless local policy dictates otherwise. Deactivate reactive NHS ester by hydrolysis before drain disposal is considered; follow regulations.

Research Use Note: For research use only. Not for human or veterinary use.

Solvent Selection

Applicability: As an NHS-activated dye, solvent choice directly affects reactivity and hydrolytic stability.

General solvent guidance (literature/best practice):

• Stock preparation: Dissolve Cy5 PEG NHS in anhydrous, oxygen-free DMSO or DMF. Typical stocks: 5–20 mM. Use amber vials; minimize headspace moisture.
• Reaction medium: For biomolecule labeling, mix a small volume of the anhydrous stock into a weakly basic, amine-free aqueous buffer to reach 5–20% v/v organic cosolvent. Carbonate/bicarbonate (pH 8.3–8.5) or phosphate buffer (pH 7.4–8.0) are commonly used. Avoid Tris, glycine, ethanolamine, or other nucleophilic buffers.
• Miscibility: DMSO and DMF are fully miscible with water, facilitating rapid dispersion of the dye and minimizing local over-labeling or aggregation.
• Dielectric/polarity (literature): DMSO ε ≈ 47; DMF ε ≈ 38 at 20°C. High polarity supports dissolution of both PEGylated dyes and salts during conjugation.
• Alternative media: For small-molecule amination, strictly anhydrous DMF/CH2Cl2 with a non-nucleophilic base (e.g., DIPEA) can be used. For bioconjugation, organic fractions >30% may denature proteins; validate carefully.

Selection tips:

• Choose DMSO for maximum solubility and low volatility; DMF can provide slightly lower viscosity for microinjections or LC loading.
• If high aqueous content is required, consider sulfonated analogs or longer PEG linkers for improved water solubility.
• Always pre-filter buffers (0.22 µm) and degas if air bubbles would interfere with optical assays.

Storage and Reconstitution

Item-specific instructions (from Product Data):

• Storage Conditions: Store at −20°C, protected from light.
• Shipped In: Ice chest + Ice pads.

Additional item-specific details (appearance, solvent content, stabilizers, reconstitution solvent/volume): Not specified for this item; refer to CoA/Spec Sheet.

General guidance (literature/best practice; not a product specification):

• Reconstitution: Dissolve immediately before use in anhydrous DMSO or DMF to prepare a concentrated stock (e.g., 5–20 mM). Vortex briefly; avoid introducing moisture. If particulates persist, sonicate gently in a room-temperature water bath for 1–2 minutes in amber vials.
• Aliquoting: Dispense single-use aliquots under dry inert gas (N2/Ar) into amber microtubes. Store aliquots at −20°C in a desiccated environment to limit NHS hydrolysis.
• Handling: Warm a sealed vial to room temperature before opening to prevent condensation ingress. Minimize exposure to ambient light; work under dim light or red light when feasible.
• Stability: Aqueous working solutions should be used promptly (within minutes to a few hours, depending on pH and temperature). Discard solutions that show significant hydrolysis or spectral shifts.
• Freeze–thaw: Avoid repeated freeze–thaw of solids or solutions; use aliquots to maintain integrity.

Always defer to the lot-specific CoA/SDS for authoritative storage, stability, and reconstitution instructions.

Structure and Identity

Item-specific identifiers available in Product Data:

• SKU: C163508
• Product Name: Cy5 PEG NHS
• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.

General structural description (literature/typical for “Cy5 PEG NHS” reagents):

• Cy5 denotes a heptamethine cyanine fluorophore (polymethine chain linking two indoleninium/benzindole-type heteroaromatics), typically bearing sulfonate or other substituents to tune solubility and spectra.
• PEG indicates a polyethylene glycol spacer (length varies by product) that enhances aqueous solubility, reduces aggregation, and increases distance from the dye to the conjugation site, improving biomolecule labeling performance.
• NHS designates an N-hydroxysuccinimide activated ester at the terminus of the PEG chain. The NHS ester selectively acylates primary amines (e.g., lysine ε-NH2, N-termini) to form stable amide bonds.

2D structure in words (literature/general):

• A cationic cyanine chromophore core (two nitrogen-containing aromatic systems) connected by a polymethine chain, appended via a linker to a PEG oligomer terminating in a carbonyl-activated succinimidyl ester. No defined stereocenters are typical; the chromophore contains conjugated double bonds susceptible to E/Z isomerism along the polymethine.

Notes:

• Exact PEG length, substitution pattern, counterions, and chromophore substituents differ among commercial Cy5 PEG NHS products and are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

Chemoselective amide formation (literature/general):

• Functional handle: The NHS ester is a classic activated carboxyl derivative enabling amide bond formation with primary amines under mild conditions. The PEG spacer imparts solubility and distance from the chromophore, useful when attaching to crowded or hydrophobic substrates.

Use cases in synthesis and materials:

• Small-molecule coupling: Install the Cy5-PEG amide onto amine-bearing scaffolds (e.g., linkers, polymers, surfaces) for fluorescence tagging or tracking in synthetic studies.
• Surface modification: Grafting onto amine-functionalized surfaces (silica, polymers, nanoparticles) to create fluorescent materials and biosensors; the PEG reduces nonspecific adsorption and aggregation.
• Orthogonality: NHS ester is orthogonal to azide–alkyne click chemistry, thiol–maleimide coupling, and SPAAC, enabling multi-functional constructs when combined with other handles on the counterpart.
• Protection/compatibility: Reactions proceed at ambient temperature and near-neutral to mildly basic pH, compatible with many protecting group schemes. Avoid excess nucleophiles that would outcompete the desired amine.

Analytical considerations:

• Monitor reaction progress by LC–MS (loss of NHS group, +PEG–Cy5 mass shift) or HPLC with fluorescence detection (Ex ~650 nm, Em ~670 nm; general cyanine settings).
• Purification: Reverse-phase HPLC or solid-phase extraction for small conjugates; desalting/ultrafiltration for macromolecules. Control light exposure throughout.

Note: Exact reactive group loading and PEG length for this item are not specified; confirm stoichiometry empirically.

Target Specificity

Not an antibody or biological targeting reagent with defined antigen/epitope specificity.

Item-specific targeting data: Not specified for this item; refer to CoA/Spec Sheet.

General note (chemoselectivity): The NHS ester reacts broadly with accessible primary amines (e.g., lysine side chains and N-termini). Labeling density and site distribution depend on amine pKa, local environment, and steric accessibility on the target molecule. For selective N-terminal labeling, adjust pH to favor lower pKa amines and/or use orthogonal strategies.