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    • Cy5 Maleimide (Non-sulfonated): High-Precision Thiol-Sele...

    2026-01-08

    Cy5 Maleimide (Non-sulfonated): High-Precision Thiol-Selective Protein Labeling Reagent

    Executive Summary: Cy5 maleimide (non-sulfonated) enables site-specific covalent labeling of cysteine residues in peptides and proteins via its maleimide group, providing precise and stable fluorescence signals for imaging (APExBIO, product page). The dye features excitation and emission maxima at 646 nm and 662 nm, respectively, with a high extinction coefficient (250,000 M-1cm-1) and a quantum yield of 0.2. Due to its low aqueous solubility, dissolution in organic solvents such as DMSO is required prior to conjugation (APExBIO). Cy5 maleimide is pivotal for generating fluorescent probes for protein tracking in advanced imaging workflows (internal source). Its robust chemistry supports reproducibility and sensitivity in high-complexity labeling protocols (internal source).

    Biological Rationale

    Protein labeling with fluorescent dyes facilitates visualization, quantification, and tracking of biomolecules in complex systems. Site-specific labeling of cysteine residues is essential because cysteines are less abundant and more nucleophilic than other amino acids, enabling selective modification without extensive off-target labeling (Chen et al., 2023). Thiol-reactive maleimide chemistry is widely adopted due to its high selectivity under mild aqueous buffering conditions (pH 6.5–7.5). Cy5 maleimide (non-sulfonated) extends these advantages by offering a bright, photostable near-infrared fluorophore compatible with advanced imaging platforms. This supports research in fields such as protein-protein interaction mapping, site-specific conjugation for targeted therapeutics, and real-time cellular imaging. The utility of Cy5 maleimide in nanomotor-driven immunotherapy and blood-brain barrier targeting has been benchmarked in recent translational research (Chen et al.).

    Mechanism of Action of Cy5 maleimide (non-sulfonated)

    Cy5 maleimide (non-sulfonated) contains a reactive maleimide moiety that forms a stable thioether bond with sulfhydryl (–SH) groups present in cysteine side chains or other thiol-containing molecules. This reaction proceeds rapidly under physiological pH (6.5–7.5) and is highly specific due to the unique nucleophilicity of thiols compared to other amino acid side chains (internal review). The cyanine-based Cy5 core provides strong absorption at 646 nm and emission at 662 nm, ensuring high sensitivity in fluorescence detection. The lack of sulfonate groups in this variant confers lower aqueous solubility but reduces nonspecific interactions, which is advantageous in certain conjugation workflows. The product is supplied as a solid and is stable for up to 24 months at –20°C when protected from light (APExBIO).

    Evidence & Benchmarks

    • Cy5 maleimide (non-sulfonated) enables selective, covalent cysteine labeling in proteins, minimizing off-target modification (Chen 2023, DOI).
    • The dye exhibits an extinction coefficient of 250,000 M-1cm-1 and quantum yield of 0.2, supporting bright, quantifiable fluorescence signals (APExBIO, product).
    • Labeling reactions proceed efficiently at pH 6.5–7.5 and room temperature, with optimal results achieved when the dye is dissolved in DMSO or ethanol (APExBIO).
    • Cy5 maleimide-conjugated probes have demonstrated high sensitivity and specificity in imaging workflows for cell tracking and protein localization (internal benchmark).
    • The dye remains stable for 24 months at –20°C in the dark and tolerates up to 3 weeks at room temperature during transportation (APExBIO).

    Applications, Limits & Misconceptions

    Applications:
    Cy5 maleimide (non-sulfonated) is widely used in:

    • Site-specific labeling of cysteine residues in recombinant proteins for fluorescence imaging (internal article).
    • Generation of fluorescent probes for biomolecule conjugation, enabling single-molecule analysis and super-resolution microscopy.
    • Tracking protein trafficking, cell viability assays, and nanomotor targeting in translational research (Chen et al.).
    • Immunotherapy and blood-brain barrier research as demonstrated in glioblastoma models.

    Limits:

    • Low aqueous solubility requires pre-dissolution in an organic co-solvent; direct addition to water leads to precipitation and inefficient labeling.
    • Non-sulfonated dyes may display increased nonspecific binding in some biological applications compared to sulfonated analogues.
    • Photobleaching can occur under prolonged exposure to intense light; storage and handling in the dark are essential.

    Common Pitfalls or Misconceptions

    • Assuming direct water solubility: Cy5 maleimide (non-sulfonated) must be dissolved in DMSO or ethanol before use.
    • Expecting reactivity with non-thiol residues: Maleimide chemistry is highly specific for sulfhydryl groups and will not efficiently label amines or hydroxyls.
    • Confusing with sulfonated variants: The non-sulfonated form differs in solubility and nonspecific binding characteristics.
    • Overestimating photostability: While robust, Cy5 dyes are susceptible to photobleaching under continuous illumination.
    • Assuming clinical/diagnostic use: The product is for research use only, not for therapeutic or diagnostic applications.

    Workflow Integration & Parameters

    For optimal labeling, dissolve Cy5 maleimide (non-sulfonated) in dry DMSO or ethanol to form a 10 mM stock solution. Add the dye to buffered protein solutions (pH 7.0 ± 0.5) in a molar ratio of 1:1 to 5:1 (dye:protein) and incubate at room temperature for 30–60 minutes, protected from light. Remove excess dye via buffer exchange or gel filtration. Store labeled conjugates at 4°C in the dark. The dye is compatible with fluorescence microscopes, plate readers, and imaging systems that support the Cy5 channel (excitation 646 nm, emission 662 nm). For detailed scenario-driven protocols and troubleshooting, refer to Scenario-Driven Best Practices for Cy5 Maleimide (Non-sulfonated), which provides practical insights beyond this article, especially for advanced cell viability and nanotechnology applications.

    This article extends prior overviews, such as Strategic Protein Labeling in Translational Research, by focusing on new evidence for workflow reproducibility and real-world benchmarking in complex biological systems.

    Conclusion & Outlook

    Cy5 maleimide (non-sulfonated) from APExBIO is a high-performance, thiol-reactive fluorescent dye for precise, site-specific protein labeling. Its robust spectral properties and conjugation efficiency make it a preferred choice for advanced imaging and translational research, including nanomotor-driven immunotherapy strategies (Chen et al., 2023). Future developments may further optimize solubility and photostability, but current evidence supports its role as a standard for covalent labeling of thiol groups in proteins and peptides. For full product details and ordering, consult the Cy5 maleimide (non-sulfonated) A8139 kit page.