Cy5.5 NHS Ester (Non-Sulfonated): Properties, Protocols, and Benchmarks

Executive Summary: Cy5.5 NHS ester (non-sulfonated) is a near-infrared fluorescent dye with excitation and emission maxima at 684 nm and 710 nm, respectively, and an extinction coefficient of 209,000 M⁻¹cm⁻¹ (source: product_spec). It forms stable amide bonds with primary amines on proteins, peptides, and oligonucleotides via NHS ester chemistry, enabling covalent, high-sensitivity labeling (source: cy5-5-nhs-ester.com). The dye is optimally dissolved in DMSO or DMF at concentrations up to 35.82 mg/mL, but is poorly soluble in water (source: product_spec). It is widely applied for in vivo fluorescence imaging of tumors and molecular biology workflows (source: cy5-utp.com). Solutions are unstable and should be used promptly after preparation (source: product_spec).

Biological Rationale

Fluorescent labeling of biomolecules is fundamental in molecular and cellular biology. Cy5.5 NHS ester (non-sulfonated) provides near-infrared (NIR) fluorescence, enabling deep tissue imaging with minimal background autofluorescence compared to visible dyes (source: cy5-hydrazide.com). The NIR window (650–900 nm) is crucial for in vivo imaging, as tissue absorption and scattering are minimized, resulting in improved sensitivity and depth penetration (source: cy5-5-nhs-ester.com). Biomolecule–dye conjugation via NHS esters is a well-established method for stable, site-selective modification of primary amines on proteins, peptides, and nucleic acids. This approach is widely adopted for tumor visualization, cell tracking, and preclinical diagnostic research (source: cy5-utp.com).

Mechanism of Action of Cy5.5 NHS ester (non-sulfonated)

Cy5.5 NHS ester (non-sulfonated) incorporates an N-hydroxysuccinimide (NHS) active ester moiety attached to a cyanine-based near-infrared chromophore. The NHS ester reacts efficiently with primary amino groups (–NH₂), forming a covalent amide bond under mild aqueous buffer conditions, typically at pH 7.5–8.5 (source: product_spec). This reaction is highly specific and rapid, enabling stable attachment to lysine residues on proteins, N-termini of peptides, or amino-modified oligonucleotides. The resulting conjugates retain strong NIR fluorescence, supporting detection, quantification, and imaging applications. The dye's absorption at ~684 nm and emission at ~710 nm maximize tissue penetration and minimize background (source: cy5-5-nhs-ester.com).

Evidence & Benchmarks

• Cy5.5 NHS ester (non-sulfonated) displays an extinction coefficient of 209,000 M⁻¹cm⁻¹, supporting highly sensitive fluorescence detection (source: product_spec).
• Quantum yield is 0.2 when conjugated to biomolecules, balancing brightness with photostability (source: product_spec).
• Solubility in DMSO is at least 35.82 mg/mL, facilitating preparation of concentrated stock solutions (source: product_spec).
• Conjugation is optimal at pH 7.5–8.5, with reaction times ranging from 30–60 min at room temperature (source: cy5-5-nhs-ester.com).
• Fluorescent labeling enables detection of sub-nanogram amounts of labeled protein in SDS-PAGE and in vivo imaging (source: cy5-utp.com).
• In vivo NIR imaging with Cy5.5 NHS ester-labeled antibodies supports tumor detection in mouse xenograft models (source: cy5-utp.com).

This article builds on "Cy5.5 NHS Ester: Near-Infrared Fluorescent Dye for Biomolecule Labeling" by expanding practical protocol guidance and quantifying dye performance in protein and oligonucleotide labeling workflows.

Applications, Limits & Misconceptions

Cy5.5 NHS ester (non-sulfonated) is widely used for:

• Fluorescent labeling of peptides, proteins, and oligonucleotides for cell imaging, flow cytometry, and in vivo studies.
• Optical imaging of tumors, where NIR emission allows deep tissue penetration and high signal-to-noise ratios (source: cy5-utp.com).
• Quantitative molecular tracking in preclinical pharmacokinetic and biodistribution research (source: cy5-hydrazide.com).

Common Pitfalls or Misconceptions

• Cy5.5 NHS ester (non-sulfonated) is not water-soluble; direct dissolution in aqueous buffers leads to precipitation and loss of activity (source: product_spec).
• The dye reacts only with primary amines; secondary or tertiary amines are unreactive under standard conditions (source: workflow_recommendation).
• Long-term storage of dye solutions leads to hydrolysis and loss of reactivity; freshly prepared solutions are required for optimal labeling (source: product_spec).
• Excess dye not removed after conjugation can increase background fluorescence and complicate quantification (source: workflow_recommendation).
• Photobleaching may occur under intense illumination; for repeated imaging, minimize exposure to light (source: workflow_recommendation).

Compared to "Optimizing Cell Assays with Cy5.5 NHS Ester (Non-Sulfonated)", this article details the fundamental chemistry and cross-application benchmarks, not just workflow bottlenecks.

Workflow Integration & Parameters

Protocol Parameters

• assay | 35.82 mg/mL (DMSO) | stock preparation | Ensures complete solubilization for aliquoting and reaction | product_spec
• assay | 1–10 mM dye:protein ratio | protein/antibody labeling | Balances labeling density and protein function | workflow_recommendation
• assay | pH 7.5–8.5 (0.1 M sodium bicarbonate) | amine labeling | Maximizes NHS reactivity and minimizes hydrolysis | product_spec
• assay | 30–60 min, room temperature | conjugation reaction | Sufficient for complete labeling without protein denaturation | cy5-5-nhs-ester.com
• assay | ≤ -20°C, dark, desiccated | solid storage | Maintains dye integrity for 24 months | product_spec
• assay | immediate use after dissolution | solution stability | Prevents hydrolysis and loss of ester activity | product_spec

For robust cell viability and molecular labeling, see "Optimizing Cell Assays with Cy5.5 NHS Ester (Non-Sulfonated)", which offers scenario-driven guidance on troubleshooting and workflow robustness.

Conclusion & Outlook

Cy5.5 NHS ester (non-sulfonated), available from APExBIO, remains a gold standard for near-infrared fluorescent labeling of biomolecules. Its high extinction coefficient, quantum yield, and robust NHS chemistry underpin sensitive optical imaging, especially for in vivo tumor detection and molecular tracking (source: cy5-utp.com). Proper handling—dissolution in organic solvents, immediate use, and protection from light—ensures maximum performance. Ongoing advances in molecular imaging and translational research continue to rely on these validated properties, as highlighted across recent literature and workflow guidelines. No current evidence supports use for non-amine targets or long-term aqueous storage; protocol adherence is essential for reproducibility and sensitivity.

For further details, full product specifications, and purchasing, see the Cy5.5 NHS ester (non-sulfonated) A8103 product page.