Cy5 PEG NHS - MW 3400 Da

Cat. No.: C163509

GRADE & PURITY MW 3400 Da

Storage

Store at -20°C,Argon charged

Shipped In

Ice chest + Ice pads

Vue d’ensemble

Cy5 labeled polyethylene glycol is a near infrared fluorescent PEG derivative that has a maximum absorption at 650 nm and emission around 670 nm. Cy5 labeled PEG derivative can be easily traced from its blue color and strong fluorescence. Additional functionality allows these fluorescent PEG derivatives to be easily conjugated to other molecule or material surfaces.

Specifications

Spécifications et pureté

MW 3400 Da

Conditions de stockage de stockage

Store at -20°C,Argon charged

Expédié en

Ice chest + Ice pads

Ce produit nécessite l'expédition en chaîne froide. Les services terrestres et autres services économiques ne sont pas disponibles.

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: Air sensitive；Hygroscopic

Application Protocols

Example protocol (general, literature-based) for labeling antibodies with Cy5 PEG NHS. Adjust to your system.

• Materials:

  • Antibody (1–5 mg/mL) in 50–100 mM sodium bicarbonate, pH 8.3–8.5 (amine‑free).
  • Cy5 PEG NHS stock prepared fresh at 5–20 mM in anhydrous DMSO.
  • Desalting column (e.g., 7 kDa cutoff) or spin filter (30–50 kDa MWCO), amber tubes.

• Steps:

  1. Exchange antibody into coupling buffer (desalt if starting from Tris/glycine).
  2. Calculate dye:protein molar ratio (e.g., 3–8:1 for IgG). In low light, add dye stock dropwise while gently mixing; keep final DMSO ≤10% v/v.
  3. Incubate 30–60 min at RT, protected from light.
  4. Quench with 20–50 mM ethanolamine, 10–15 min.
  5. Purify to remove free dye using desalting column or spin filtration into storage buffer (e.g., PBS).
  6. Determine degree of labeling by UV‑Vis using protein A280 with dye correction factor and dye Aλmax (~650 nm, literature).

• Notes:

  • Avoid primary amine buffers and excessive thiols during coupling.
  • For peptides/small molecules, similar steps apply; use DIPEA in anhydrous DMF/DMSO and purify by HPLC.

This is a general guideline, not item‑specific. Optimize empirically.

Biological Roles

• Intrinsic biological role: None. Cy5 PEG NHS is a synthetic fluorophore derivative intended as a chemical labeling reagent.

• Biochemical utility (general/literature):

  • Enables far‑red fluorescence tagging of biomolecules via amide formation at primary amines, supporting detection in low‑autofluorescence windows.
  • Far‑red Cy5 emission (~660–670 nm, literature) reduces background from biological samples, improving sensitivity for microscopy, Western blotting, and flow cytometry (research use only).
  • PEG linker can reduce nonspecific interactions and steric hindrance, often improving labeling uniformity and preserving biomolecular function.

• Photophysical notes (literature):

  • Typical excitation ~640–650 nm; emission ~660–670 nm; compatible with common 633/635/640 nm laser lines and Cy5 filter sets.
  • Susceptible to photobleaching and singlet oxygen–mediated degradation; protect from light and consider antifade agents when appropriate.

• Biological pathways: Not applicable; the compound is not a metabolite or signaling molecule.

Research Use Note: For research use only (per Product Data).

Buffer Applications

This product is not a buffer reagent per se; however, buffer choice is critical for successful NHS‑ester conjugations (general guidance).

• Recommended amine‑free coupling buffers (literature):

  • 50–100 mM sodium bicarbonate/carbonate, pH 8.3–8.8.
  • 50–100 mM sodium phosphate, pH 7.8–8.2 (slower than bicarbonate but gentler for sensitive proteins).
  • Optional 100–150 mM NaCl to maintain ionic strength and solubility.

• Buffers to avoid during coupling:

  • Tris, glycine, ammonium salts, or other primary amines (compete with labeling).
  • High concentrations of thiols (e.g., DTT, β‑ME) may reduce or quench fluorescence and can react with activated esters.

• Practical tips:

  • Desalt proteins into amine‑free buffer before adding dye stock (DMSO/DMF). Keep final organic solvent content typically ≤10% v/v.
  • Maintain pH stability throughout the reaction; CO2 absorption can acidify bicarbonate buffers — prepare fresh.
  • After labeling, quench residual NHS ester with ethanolamine or Tris, then exchange into storage buffer.

All parameters above are general/literature guidance; optimize empirically for your target.

Green Alternatives

Greener handling focuses on solvent choice and process efficiency; the dye core is fixed by application needs.

• Solvent considerations (general/literature):

  • Prefer anhydrous DMSO over DMF/NMP when feasible due to a more favorable EHS profile and lower regulatory burden.
  • Minimize solvent volumes by preparing concentrated stocks and using micro-volume conjugations.
  • Employ aqueous‑compatible variants (e.g., sulfonated Cy5) to reduce aprotic solvent content when compatible with the biomolecule; note that NHS esters still hydrolyze in water.

• Energy and waste reduction:

  • Perform reactions at ambient temperature and near‑neutral to mildly basic pH to avoid cooling/heating energy loads.
  • Use rapid desalting or spin filtration instead of lengthy chromatographic purifications when suitable.

• Comparative snapshot (general guide):

  | Aspect | DMSO | DMF | NMP | |---|---|---|---| | EHS profile | Better | Moderate concerns | Greater concerns | | Volatility/odor | Low | Moderate | Low | | Typical use | First choice for stocks | Alternative | Alternative |

Tradeoffs: DMSO can oxidize sulfur‑sensitive targets and may permeabilize cells; DMF/NMP offer lower viscosity but higher EHS concerns. Choose based on target compatibility and lab policy.

Pharmaceutical Uses

This reagent is intended for research use only and is not an approved pharmaceutical ingredient.

• Possible roles in pharma R&D workflows (general):

  • Preparation of fluorescently labeled antibodies, peptides, or drug conjugates for analytical method development, target engagement studies, or biodistribution experiments in preclinical research.
  • In‑process visualization, tracer studies, and assay development within QC/analytical labs.

• Excipient/pharmacopeial status: Not specified for this item; refer to CoA/Spec Sheet. No pharmacopeial monograph is implied.

• Manufacturing considerations (R&D context):

  • PEG linker may enhance aqueous handling of labeled constructs, aiding formulation screening.
  • Ensure removal of residual DMF/DMSO and unreacted dye by validated purification before any use beyond discovery research.

No medical or clinical claims are made or implied.

Physical Properties

Item-specific physical constants are not provided. Where useful, literature/typical properties of Cy5-type NHS PEG esters are summarized for context.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet (varies with PEG length and sulfonation state).
• Solubility (general/literature):
  • Highly soluble in anhydrous polar aprotic solvents (DMSO, DMF, NMP).
  • Variable solubility in aqueous buffers; PEG improves dispersibility. NHS ester hydrolyzes in water over time.
• Spectral properties (literature, Cy5-class fluorophore):
  • Typical λmax(abs) ~640–650 nm; λmax(em) ~660–670 nm in aqueous media, dependent on exact structure and environment.
  • High molar absorptivity and moderate-to-high quantum yield; can be sensitive to polarity and aggregation.
• Partitioning (general): PEGylation reduces hydrophobic aggregation relative to non‑PEG Cy5; logP is strongly structure- and counterion-dependent (not specified for this item).
• Melting point, boiling point, density, refractive index: Not specified for this item; refer to CoA/Spec Sheet.

Important: Treat spectral numbers as literature guidance only; verify with the CoA/Spec Sheet for this specific catalog item before use in quantitative work.

Quality and Grades

• Grade/Purity (item-specific): Not specified for this item; refer to CoA/Spec Sheet.

• What grade typically implies for amine‑reactive dyes (general guidance):

  • Research grade: Suitable for most biochemical labeling; key parameters include purity by HPLC, identity by MS, residual solvents, and dye content. Low hydrolyzed NHS content and minimal free acid are important for efficient conjugation.
  • Chromatography grade (if specified): Low UV‑absorbing impurities across the visible range to minimize background.

• Stabilizers/additives: Not specified for this item; refer to CoA/Spec Sheet. Many NHS esters are supplied without stabilizers and rely on dry, inert storage.

• Quality considerations specific to Cy5 PEG NHS (practical notes):

  • PEG length distribution can broaden by ±1 EO unit depending on synthesis; this can slightly broaden MS and HPLC profiles (literature). Verify acceptance criteria on the Spec Sheet.
  • Degree of hydrolysis during storage/transport affects labeling efficiency. Confirm active ester content (e.g., by test coupling or HPLC) if critical.
  • Counterions (e.g., TFA, chloride, sodium) influence solubility and spectral baseline; consult CoA for the supplied form.

Always consult the item’s CoA/Spec Sheet for definitive specifications, test methods, and acceptance criteria.

Reaction and Applications

This reagent is an NHS ester of a Cy5 fluorophore bearing a PEG spacer, designed for efficient amide‑bond formation with primary amines.

• Primary application domains (general/literature):

  • Protein and peptide labeling at lysine side chains or N‑termini for fluorescence detection, imaging, or assay readouts.
  • Small‑molecule and surface functionalization (e.g., amine‑terminated polymers, nanoparticles, and resins).
  • Probe and tracer synthesis for biochemical assays, flow cytometry, and microscopy (research use only).

• Reaction chemistry (mechanistic overview):

  • NHS ester undergoes nucleophilic acyl substitution by primary amines to give an amide and N‑hydroxysuccinimide byproduct. Reaction rate increases with deprotonation of the amine (pH ~8–8.8 in aqueous buffers) and in dry aprotic media with non-nucleophilic base for small molecules.

• Practical tips (general):

  • Work under low light; minimize water exposure. Prepare fresh, anhydrous stock (e.g., in DMSO), then add to buffered target solution.
  • Avoid competing nucleophiles (e.g., Tris, glycine, azide in high concentrations) during coupling.
  • Control degree of labeling (DOL) via dye:protein molar ratio, reaction time (typically 30–120 min at RT), and pH.
  • Remove excess/unreacted dye by desalting columns, ultrafiltration, or HPLC depending on application.

• PEG spacer advantages:

  • Improves solubility, reduces aggregation and self‑quenching, and can enhance labeling homogeneity on crowded biomolecular surfaces (literature observations).

Reaction Conditions

General literature guidance for NHS‑ester conjugation; adjust to your system.

• Protein/peptide labeling (aqueous):

  • Buffer: 50–100 mM sodium bicarbonate (pH 8.3–8.8) or phosphate (pH 7.8–8.2), amine‑free, optional 100–150 mM NaCl.
  • Substrate concentration: 0.5–5 mg/mL protein typical.
  • Dye addition: Add freshly prepared dye stock in anhydrous DMSO/DMF; keep final organic ≤10% v/v.
  • Stoichiometry: 2–10 molar equivalents of dye per accessible amine (or per protein, depending on desired DOL).
  • Temperature/time: RT, 30–120 min in the dark; gentle mixing.
  • Quench: 10–50 mM ethanolamine or Tris after desired conversion, then purify.

• Small‑molecule coupling (aprotic):

  • Solvent: Anhydrous DMF or DMSO.
  • Base: 1–3 equiv of non‑nucleophilic base (e.g., DIPEA, Hünig’s base) to deprotonate amine.
  • Temperature: 0–25°C; typical times 0.5–4 h.
  • Workup: Dilute, aqueous wash to remove NHS; purify by chromatography.

• Analytical monitoring:

  • UV‑Vis at Cy5 λmax (~640–650 nm) to track consumption/integration (literature values).
  • LC/MS or SDS‑PAGE with in‑gel fluorescence for proteins.

These ranges are literature norms; confirm on small scale before scale‑up.

Safety and Handling

Safety details specific to this item are not provided in the Product Data; consult the SDS for authoritative information.

• GHS/SDS information:

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

• General hazards and precautions for NHS-activated cyanine dyes (literature/good practice):

  • NHS esters are moisture‑sensitive; hydrolysis reduces activity. Handle under dry, inert atmosphere (argon or nitrogen) and minimize exposure to ambient humidity.
  • May cause skin/eye irritation; avoid inhalation/ingestion. Use appropriate PPE (lab coat, safety glasses, nitrile gloves) and work in a fume hood when weighing and dissolving.
  • Photosensitive: Protect from light to avoid photobleaching and decomposition.

• Incompatibilities (general):

  • Aqueous bases and nucleophiles (e.g., primary amines, hydroxide) accelerate NHS ester hydrolysis/consumption.
  • Avoid amine‑containing buffers (Tris, glycine) during conjugation setup.

• First aid overview (general):

  • Skin/eye contact: Rinse with water for ≥15 minutes; remove contaminated clothing; seek medical attention as needed.
  • Inhalation: Move to fresh air; seek medical attention if symptoms persist.
  • Ingestion: Rinse mouth; seek medical attention.

• Fire and spill (general):

  • Many organic dyes are combustible; use CO2, dry chemical, or foam. For spills, avoid dust formation, collect with inert absorbent, and dispose per local regulations.

Storage per Product Data: Store at −20°C, argon charged; protect from moisture and light. Shipped in ice chest with ice pads.

Solvent Selection

Cy5 PEG NHS is typically handled in dry, polar aprotic solvents to preserve NHS ester activity and achieve high stock concentrations.

• Polarity/miscibility (general):

  • Preferred: Anhydrous DMSO, DMF, or NMP — dissolve readily and are miscible with water for subsequent dilution into labeling buffers.
  • Avoid initial dissolution in water or alcohols; NHS hydrolyzes, reducing coupling efficiency.

• Choosing among common options (general guidance):

  • DMSO: Low toxicity profile relative to DMF/NMP; excellent solvency; viscous at low temperatures. Widely used for bioconjugation stocks.
  • DMF: Excellent solvency; lower viscosity; handle with care due to toxicity concerns; ensure anhydrous grade.
  • NMP: Strong solvency and stability; higher boiling point can complicate removal.

• Aqueous phase considerations:

  • For conjugation, the dye stock in DMSO/DMF is added to a pH 8.0–8.8 amine‑free buffer (e.g., sodium bicarbonate or phosphate). Maintain final organic content typically ≤10% v/v to preserve biomolecule structure (literature guidance).
  • Avoid Tris, glycine, or other primary amine buffers during coupling.

• Quick comparison (general):

  • Fast dissolution and ease-of-use: DMSO > DMF ≈ NMP
  • Worker safety profile: DMSO > DMF > NMP (literature consensus)

Always verify compatibility with your target biomolecule and validate final solvent composition empirically.

Storage and Reconstitution

• Storage conditions (from Product Data):

  • Store at −20°C, argon charged.
  • Shipped in ice chest with ice pads.

• Additional handling guidance (general best practice for NHS‑ester dyes):

  • Protect from moisture and light at all times; keep container tightly closed. Consider secondary containment with desiccant in the dark.
  • Allow vial to warm to room temperature in a desiccator before opening to prevent condensation.

• Reconstitution (general):

  • Prepare fresh stock solutions in anhydrous DMSO or DMF immediately before use. Typical working stocks are in the millimolar range; choose concentration based on application. Specific recommended concentration: Not specified for this item; refer to CoA/Spec Sheet.
  • After dissolution, aliquot into amber microvials under dry inert gas to minimize headspace moisture; freeze at −20°C or below.

• Stability (general):

  • NHS esters slowly hydrolyze even when frozen if moisture is present; minimize freeze–thaw cycles by aliquoting. Avoid repeated warming.
  • In aqueous buffers, use promptly; working solutions are typically used within hours at RT. Exact limits are application‑dependent and not specified for this item.

Always refer to the item’s CoA/Spec Sheet and SDS for definitive instructions and stability data.

Structure and Identity

Brief overview: Cy5 PEG NHS is an amine‑reactive fluorescent labeling reagent consisting of a Cy5 (heptamethine cyanine-5) fluorophore connected via a polyethylene glycol (PEG) spacer to an N‑hydroxysuccinimide (NHS) ester.

• Item-specific identifiers (from Product Data):

  • CAS: 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.
  • InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES: Not specified for this item; refer to CoA/Spec Sheet.

• Structural features (general/literature description of this class):

  • Fluorophore: Cy5 indocarbocyanine core with a conjugated heptamethine polymethine chain linking two indole/benzindole units; typically provides far‑red absorption/emission.
  • Linker: A PEG chain (length not specified for this item) that enhances aqueous solubility and reduces aggregation/steric hindrance.
  • Reactive group: Terminal NHS ester enabling acylation of primary amines to form stable amide bonds.
  • 2D description in words: An extended, planar cyanine chromophore connected through a flexible, ether‑rich PEG chain terminating in a five‑membered succinimidyl carbonate‑like ring (NHS) poised for nucleophilic acyl substitution by –NH2.

• Stereochemistry: No defined stereocenters in the Cy5 core; PEG is achiral; NHS ester is achiral.

Note: Exact substitution pattern, counterions, and PEG length are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

As an activated ester, Cy5 PEG NHS is a convenient handle for installing a far‑red fluorophore onto amine‑bearing substrates.

• Functional group reactivity (general/literature):

  • Highly selective toward primary aliphatic amines; secondary amines react more slowly. Minimal reaction with alcohols/thiols under neutral conditions.
  • Forms robust amide linkages under mild conditions; byproduct is N‑hydroxysuccinimide.

• Retrosynthetic value:

  • Enables late‑stage diversification of complex molecules (peptides, small molecules, surfaces) without protecting groups when amines are uniquely accessible.
  • PEG spacer provides distance from the chromophore, mitigating photophysical perturbation and steric occlusion.

• Representative transformations:

  • Labeling of lysine‑containing peptides/proteins in aqueous buffer (pH ~8–8.8).
  • Coupling to amine‑terminated polymers (e.g., PEG‑NH2, PAMAM) or nanoparticles for imaging probes.
  • Small‑molecule tagging in anhydrous DMF/DMSO with non‑nucleophilic base (e.g., DIPEA) at 0–25°C.

• Practical notes:

  • Control stoichiometry and time to tune degree of labeling and avoid over‑modification that can impair function.
  • Purify conjugates by SEC, spin filtration, or RP‑HPLC depending on size/hydrophobicity.

Target Specificity

Not applicable. This product is a chemical labeling reagent (Cy5 PEG NHS) and is not an antibody, enzyme, or biologic with inherent target specificity. Conjugation confers fluorescence to the user’s chosen amine‑containing target. No clone, isotype, antigen, or species reactivity applies.