ATTO 565 biotin , CAS No.A1452696

CAS: A1452696 Cat. No.: A1452696 Formula: C₄₆H₅₇ClN₆O₁₀S Peso molecolare: 921.50

Storage

Protected from light,Store at -20°C,Desiccated

Shipped In

Ice chest + Ice pads

Panoramica

ATTO 565 biotin is a biotin derivative of ATTO 565 with maximum excitation/emission wavelengths: 564/590 nm.

Specifications

Condizioni di conservazione di stoccaggio

Protected from light,Store at -20°C,Desiccated

Spedito in

Ice chest + Ice pads

Questo prodotto richiede spedizione a catena fredda. I servizi di terra e altri servizi economici non sono disponibili.

Nomi e identificatori

• Peso molecolare: 921.50

Application Protocols

• Important note
  • No item-specific validated protocols or dilutions are provided for this SKU. The following are general, literature-based guidelines for fluorescent biotin probes and should be optimized by the user.
• General staining/detection on fixed cells or tissue
  1. Prepare a 1–10 mM stock in anhydrous DMSO. Protect from light.
  2. Block sample with 1–5% BSA or appropriate blocking buffer for 30–60 min.
  3. If using a streptavidin-anchored detector, incubate sample with the streptavidin conjugate first, wash, then apply ATTO 565 biotin (e.g., 10–200 nM in PBS + 0.1% BSA) for 15–60 min at room temperature in the dark.
  4. Wash 3× with PBS/TBS + 0.05% Tween-20. Mount with anti-fade medium.
• Blot/ELISA detection
  • After immobilizing/biotinylating the analyte and blocking, apply streptavidin (unlabeled or functionalized), then detect with ATTO 565 biotin or, alternatively, detect with fluorescent streptavidin if the analyte is biotinylated. Typical probe concentration 50–500 nM; adjust based on background.
• Bead-based capture/flow cytometry
  • For streptavidin-coated beads, incubate with ATTO 565 biotin at 10–200 nM for 10–30 min, wash thoroughly, and analyze with a 561 nm laser and 585/42–610/20 nm emission filter.
• Controls and QA
  • Include no-probe, no-streptavidin, and free-biotin competition controls. Record illumination settings to track photobleaching and ensure reproducibility.

Biological Roles

• Biotin biochemistry (literature, background)
  • Biotin (vitamin B7/H) is a covalently attached cofactor for carboxylase enzymes, cycling CO2 in metabolic pathways such as fatty acid synthesis and gluconeogenesis. Endogenous biotinylation occurs via biotin ligase (holocarboxylase synthetase) on specific lysine residues.
• Streptavidin/avidin binding (literature)
  • The avidin family (avidin, streptavidin, NeutrAvidin) binds biotin with sub-femtomolar affinity, driven by a deep hydrophobic pocket and extensive hydrogen bonding. Association is rapid and highly specific under near-neutral pH and physiological ionic strength.
• Relevance to this product
  • ATTO 565 biotin leverages the biotin–streptavidin interaction to deliver a bright orange-red fluorescent signal wherever streptavidin (or biotin-binding proteins) are localized. This enables two-step detection: either a biotinylated primary reagent detected by fluorescent streptavidin, or a fluorescent biotin detected by avidinylated targets/surfaces.
• Considerations in biological samples
  • Endogenous biotinylated proteins can contribute background; pre-blocking strategies or biotin depletion steps can improve specificity.
  • Biotin binding is essentially irreversible under native conditions but can be disrupted by harsh conditions (e.g., hot SDS, high concentrations of chaotropes). Plan elution strategies accordingly for capture/release workflows.
• No clinical/therapeutic claims
  • This product is intended solely for laboratory research applications involving detection, imaging, and assay development; it is not validated for diagnostic or therapeutic use.

Buffer Applications

• Applicability
  • This product is a fluorescent probe, not a buffering reagent. Buffer selection focuses on maintaining binding efficiency and fluorescence, not on pH control by the reagent itself.
• Recommended buffer systems (general guidance)
  • PBS or TBS at pH 7.2–7.6 provide optimal ionic strength and pH for streptavidin–biotin binding and for preserving ATTO 565 fluorescence.
  • Avoid strong reducing agents at high concentration (e.g., DTT >10 mM) if using redox-sensitive co-stains; they typically do not affect biotin binding but may influence other assay components.
  • Include protein/blocking additives to minimize nonspecific adsorption: 0.1–1% BSA or casein in incubation buffers; 0.01–0.1% Tween-20 for wash buffers.
• Practical tips
  • Filter buffers (0.2 µm) and degas as needed for imaging applications to reduce background scattering and bubble formation.
  • Maintain low light exposure during incubations and washes. Use amber tubes and foil-wrap plates when feasible.
• Compatibility notes
  • Chelators (e.g., EDTA) and common salts (NaCl, KCl) are compatible with biotin–streptavidin binding at typical concentrations.
  • High concentrations of free biotin in serum-containing media can compete with binding; for sensitive assays, use serum-free buffers or implement biotin-blocking steps.

Green Alternatives

• Environmental considerations (general)
  • The primary environmental load arises from organic solvents (DMSO/DMF) and single-use plastics in bioassays rather than the microgram quantities of dye. Minimizing hazardous solvent use and dye waste is the main opportunity.
• Greener practice suggestions
  • Prefer aqueous buffers for final working solutions; restrict DMSO stock concentrations to the minimum needed for solubility.
  • Replace DMF with DMSO or glycerol/water co-solvent when feasible; both offer lower volatility and more favorable hazard profiles.
  • Implement microvolume staining to reduce total reagent consumption; reuse washing buffers where cross-contamination is not a risk.
• Alternative fluorophores or formats
  • If instrument filters allow, consider water-enhanced dyes that dissolve directly in PBS, reducing organic co-solvent content. Evaluate photostability and brightness trade-offs versus ATTO 565.
• Small comparison (general, literature)
  • Attribute | ATTO 565 biotin | Alexa Fluor 555-biotin | Cy3-biotin
  • Brightness (relative) | High | High | Moderate–High
  • Photostability | High | High | Moderate
  • Solubility | Often DMSO stock | Often DMSO stock | DMSO stock
  • Green impact | Similar; dominated by solvent handling | Similar | Similar
• Waste reduction
  • Aliquot stocks to avoid freeze–thaw losses; prepare only the amount needed per experiment to minimize expired waste.
  • Collect dye-containing waste separately to enable appropriate disposal and prevent dye release to aqueous streams.

Pharmaceutical Uses

• Scope
  • ATTO 565 biotin is supplied for research use only. It is not formulated, qualified, or intended for clinical diagnostic or therapeutic applications.
• Formulation/excipient context (general information)
  • In pharmaceutical development research, biotinylated fluorophores are occasionally used as in vitro analytical probes to study bioconjugation efficiency, receptor–ligand interactions on streptavidin-functionalized platforms, or to QC biotinylated surfaces. These are internal R&D utilities rather than drug product components.
• Regulatory status
  • No pharmacopeial monograph is associated with this conjugate. Any GMP-related use would require independent qualification, impurity profiling, and validation specific to the intended process.
• Practical considerations for R&D groups
  • Define acceptance criteria for spectral identity (λmax, ε), purity by HPLC, and residual solvents. Establish robust storage and handling SOPs to prevent light/moisture degradation.
  • For method transfer or cross-site comparability, include dye-labeled reference standards and standardized illumination/detector settings to control for photophysics variability.
• Disposal and documentation
  • Treat as a laboratory chemical; document use and disposal per institutional policies and applicable regulations.

Physical Properties

• Item-specific physicochemical specs
  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.
  • Purity/grade: Not specified for this item; refer to CoA/Spec Sheet.
  • Melting/boiling points, density, refractive index: Not specified for this item; refer to CoA/Spec Sheet.
  • Solubility: Not specified for this item; refer to CoA/Spec Sheet.
• Spectroscopic/photophysical properties (literature, typical for ATTO 565 family; not item-specific specifications)
  • Absorption maximum (λmax abs): ~563 nm (aqueous/MeOH, literature)
  • Emission maximum (λmax em): ~592 nm (literature)
  • Extinction coefficient (ε): ~1.2 × 10^5 M⁻¹·cm⁻¹ (literature)
  • Quantum yield (ΦF): up to ~0.9 in suitable media (literature)
  • Typical Stokes shift: ~25–30 nm (literature)
• Solubility/handling characteristics (general guidance)
  • Many dye–biotin conjugates are readily dissolved in anhydrous DMSO to prepare concentrated stocks (1–10 mM), and can be diluted into aqueous buffers containing 0.01–0.1% surfactant (e.g., Tween-20) or 0.1% BSA to minimize nonspecific adsorption.
  • The biotin motif is hydrophobic; aqueous solubility of the neat conjugate varies with linker/charge pattern on the dye. Test a small aliquot for clarity upon dilution.
• Stability (general)
  • Light-sensitive; photobleaches upon strong illumination and in the presence of oxygen/singlet oxygen generators. Protect from light during all manipulations.
  • Avoid prolonged exposure to elevated temperature and moisture to preserve dye integrity.

Quality and Grades

• Item-specific grade/purity
  • Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• What “research grade” fluorescent biotin probes typically imply
  • Produced for laboratory use in imaging, assay development, and biochemical detection, not for diagnostic or therapeutic applications.
  • Purity is commonly assessed by HPLC/UPLC and identity by MS; spectral quality (λmax/ε) may be confirmed against internal standards. For this specific item, please confirm acceptance criteria on the CoA.
• Stabilizers and formulation
  • Many fluorophores are supplied neat (lyophilized) without stabilizers or as films; some are formulated with trace desiccants. No stabilizers are specified for this item; refer to the Spec Sheet for any additives.
• Performance considerations for biotinylated dyes
  • Lot-to-lot consistency in dye loading and linker chemistry ensures predictable brightness and binding. Review provided spectral scans and HPLC traces when available.
  • For quantitative work, determine the dye concentration by absorbance using the literature ε for ATTO 565 and verify by fluorometric standard curves; adjust for solvent- and pathlength-dependent effects.
• Documentation
  • CoA/Spec Sheet should be consulted for: purity by HPLC, residual solvents, counterions/salts, water content, and recommended extinction coefficient to use for concentration determinations.

Reaction and Applications

• Core application domain
  • Fluorescent labeling via the biotin–avidin/streptavidin/NeutrAvidin interaction for microscopy, flow cytometry, blotting, ELISA, nucleic acid detection, and bead-based assays.
• Binding chemistry (general/literature)
  • Biotin binds tetrameric streptavidin with extremely high affinity (Kd ~10⁻¹⁴–10⁻¹⁵ M, literature), enabling near-irreversible complexation under physiological conditions. This permits modular, two-step labeling: (1) biotinylated probe targets analyte, (2) streptavidin (labeled or unlabeled) bridges to reporters or surfaces.
• Optical channel usage
  • ATTO 565 is excited efficiently by 532–568 nm light and emits around 590 nm (literature), ideal for confocal systems with 561 nm lasers and standard TRITC/Cy3 filter sets.
• Practical considerations
  • Block free biotin in samples (e.g., use serum-free conditions or perform avidin/biotin blocking) to minimize background when probing endogenous biotinylated proteins (carboxylases).
  • Optimize probe concentration by titration (typical working range 1–200 nM in imaging; 10–1000 nM in blot/ELISA), balancing brightness and nonspecific binding. Validate for your matrix.
  • Use low-light conditions during incubations; include oxygen scavengers or anti-fade reagents in imaging media to reduce photobleaching.
• Extended applications
  • Super-resolution compatible workflows where bright, photostable orange-emitting labels are advantageous (validate switching/compatibility).
  • Surface patterning and capture on streptavidin-coated chips, nanoparticles, or magnetic beads for enrichment and detection.

Reaction Conditions

• Binding/assay conditions (general guidance; literature-informed)
  • Streptavidin–biotin complexation proceeds efficiently at pH 6.5–8.5 in PBS/TBS, 20–200 mM salt, ambient temperature. Incubation times of 5–30 min are often sufficient for surface-bound systems; 30–60 min for tissue/cell staining.
  • Working concentrations typically range from 1–200 nM for microscopy and 10–1000 nM for blotting/ELISA, depending on surface density and detection sensitivity. Titrate to minimize background.
• Imaging considerations
  • Excite with 532–568 nm sources (e.g., 561 nm laser). Collect emission with 580/30–605/40 nm or TRITC/Cy3 filter sets (literature). Use anti-fade mounting media to enhance photostability.
• Elution/disruption (if capture on streptavidin surfaces)
  • The complex is extremely stable; harsh conditions (e.g., 6–8 M guanidine HCl, hot SDS-PAGE sample buffer, or excess free biotin at elevated temperature over extended periods) may partially release captured material. Plan for single-use capture when possible.
• Compatibility
  • Common buffers, salts, and mild detergents are compatible. Avoid high free-biotin backgrounds and minimize exposure to strong oxidants or UV to preserve fluorescence.
• Note
  • These conditions are provided as general literature-based guidance for ATTO 565–type biotin probes. They are not item-specific specifications; optimize empirically for your system.

Safety and Handling

• GHS classification and hazard statements
  • Signal word: Not specified for this item; refer to SDS.
  • GHS pictograms / hazard classes / H-statements: Not specified for this item; refer to SDS.
• General laboratory precautions (best practice for fluorescent bioconjugates)
  • Handle in a well-ventilated area wearing appropriate PPE: lab coat, safety glasses, and disposable nitrile gloves. Avoid inhalation of powders/aerosols and skin contact.
  • Dyes can stain skin and labware; work over bench paper and clean spills promptly with suitable solvent while protecting from light.
• Light and moisture sensitivity
  • Keep the material protected from ambient light during weighing, dissolution, aliquoting, and storage to minimize photobleaching and photooxidation.
  • Maintain desiccation; avoid repeated opening to humid air to reduce hydrolysis or aggregation.
• Incompatibilities (general)
  • Strong oxidizers, strong acids/bases may degrade the fluorophore or the biotin amide linkage. Sodium azide at high concentration can quench some fluorescence signals; consider compatibility with downstream assays.
• First-aid overview (non-authoritative; consult SDS)
  • Skin/eye contact: rinse with water for several minutes; remove contaminated clothing. Seek medical attention if irritation persists.
  • Ingestion/inhalation: move to fresh air; rinse mouth with water; obtain medical advice.
• Waste disposal
  • Collect dye-containing solutions as organic/halogen-free waste per institutional and local regulations. Decontaminate glassware with appropriate solvent rinses before aqueous washing.

Solvent Selection

• Polarity/solubility profile (general for dye–biotin conjugates)
  • Highly soluble in anhydrous DMSO and DMF; limited to moderate solubility in alcohols (MeOH/EtOH). Aqueous solubility varies; inclusion of 0.01–0.1% nonionic surfactant or 0.1% BSA can improve performance in buffers.
  • Avoid prolonged storage in dilute aqueous solutions; prepare fresh working dilutions from concentrated DMSO stocks.
• Typical working buffers
  • PBS, TBS, or HEPES-buffered saline at pH 7.2–7.6 are commonly used for streptavidin-based detections. Additives: 0.05–0.1% Tween-20 for washing steps; 1–5% BSA or casein to block nonspecific sites.
• When to choose ATTO 565 biotin vs alternatives
  • Choose ATTO 565 biotin when orange-red emission is desired with green/yellow excitation (e.g., 561–568 nm laser lines). It offers low spectral overlap with fluorescein and good separation from far-red channels.
  • Alternatives: biotin conjugates of Alexa Fluor 555/568, Cy3, or TRITC for similar spectral windows; select based on instrument filters, required brightness, and photostability.
• Practical tips
  • Use anhydrous, oxygen-minimized stock solutions to improve stability. Filter aqueous working solutions (0.2 µm) if particulate forms.
  • Avoid high concentrations of primary amines (e.g., Tris) only if working with reactive esters; for this product as a finished biotin–dye conjugate, standard buffering is acceptable.

Storage and Reconstitution

• Item-specific storage and shipping
  • Storage conditions: Protected from light, Store at -20°C, Desiccated (per Product Data).
  • Shipped in: Ice chest + Ice pads (per Product Data).
• Reconstitution (general guidance)
  • Dissolve the material in anhydrous DMSO to prepare a 1–10 mM stock solution. Mix gently to avoid foaming. If particulate remains, briefly sonicate or warm to room temperature in the dark.
  • For aqueous applications, dilute the DMSO stock into PBS/TBS containing 0.1% BSA or 0.01–0.1% Tween-20 to minimize adsorption losses. Keep final DMSO ≤1% v/v where biological compatibility is required.
• Aliquoting and handling
  • Dispense single-use aliquots into amber or foil-wrapped microtubes to avoid repeated freeze–thaw. Purge headspace with inert gas if possible, cap tightly, and keep desiccated.
  • Working solutions in buffer are best prepared fresh and used the same day. If storage is necessary, keep at 4°C protected from light and use within 24–48 h.
• Stability notes
  • Avoid exposure to light, moisture, and elevated temperatures. Do not store dilute aqueous solutions for extended periods.
• Specifications not provided
  • Grade/purity, counterion, and exact solvent recommendations: Not specified for this item; refer to CoA/Spec Sheet.
• Research use
  • For research use only (per Product Data).

Structure and Identity

• Item-specific identifiers
  • CAS: A1452696 (catalog placeholder)
  • 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.
• Structural description (general/literature)
  • ATTO 565 biotin is a conjugate of a xanthene/rhodamine-class fluorophore (ATTO 565) covalently linked to biotin via a short spacer. The dye core contains a substituted xanthene scaffold with delocalized cationic/zwitterionic character, giving strong absorption in the green-yellow region and orange-red emission. The biotin moiety is a bicyclic ureido-thiophane heterocycle bearing a valeric acid side chain; in conjugates it is typically amide-coupled to a linker attached to the dye.
  • 2D features (general): one fused tricyclic xanthene ring system with amino/alkyl substituents; an amide-linked biotin heterobicycle (imidazolidone fused to tetrahydrothiophene). No defined stereocenters in the dye core; biotin contains natural stereocenters (biologically active configuration).
• Identity notes
  • “ATTO 565” specifies the photophysical family (absorption/emission maxima around 560/590 nm, literature), while “biotin” specifies high-affinity binding capability to avidin/streptavidin/NeutrAvidin systems.
  • This product is intended for research use as a fluorescent biotin probe; verify exact substitution pattern and linker length on the product CoA/Spec Sheet before method validation.

Synthetic Utility

• Applicability
  • As a finished biotin–dye conjugate, this product is primarily a detection probe rather than a building block for multistep organic synthesis.
• Utility in bioconjugation workflows (general)
  • Serves as a modular reporter that couples to streptavidin/avidin-functionalized biomolecules, beads, or surfaces, enabling rapid assembly of fluorescent complexes without covalent chemistry.
  • Facilitates orthogonal labeling strategies: combine with other spectral channels (e.g., far-red dyes) for multiplexed detection.
• Retrosynthetic considerations
  • Biotin–dye constructs are commonly made via amide formation between activated dye derivatives (e.g., NHS esters) and biotin/linker amines; while not the role of this product, understanding this enables troubleshooting stability (amide hydrolysis) and optimizing solvent choices.
• Functional group compatibility (use context)
  • Stable to near-neutral aqueous buffers, moderate salts, and typical blocking proteins. Sensitive to strong acids/bases and strong oxidants that can cleave amides or bleach xanthene dyes.
• Analytical characterization (recommended for users developing new assays)
  • Confirm integrity by absorbance/fluorescence spectroscopy (expected ATTO 565-like spectra, literature), HPLC to monitor single major species, and, when available, MS profiling for the intact conjugate.

Target Specificity

• Item-specific antigen/clone/isotype
  • Not applicable; this product is not an antibody and has no clone or isotype.
• Functional binding target (literature/general)
  • Biotin moiety binds with high specificity and affinity to the biotin-binding sites of streptavidin, avidin, and NeutrAvidin. The interaction is orthogonal to most biological ligands, enabling selective targeting of avidinylated reagents or streptavidin-coated substrates.
• Cross-reactivity considerations
  • Endogenous biotinylated proteins in cells and tissues can present binding sites and contribute to background. Employ biotin/avidin blocking steps when visualizing targets in complex samples.
• Practical implication
  • Specificity is dictated by the presence and accessibility of biotin-binding proteins or surfaces; spatial targeting in biological experiments is typically achieved by pre-localizing streptavidin conjugates (e.g., streptavidin–antibody constructs) or by employing biotinylated primaries detected with streptavidin while reserving this fluorescent biotin for counter-binding schemes.