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Application Protocols
The following are general, literature-based workflows for SPAAC labeling with DBCO-dPEG12-carboxyfluorescein. They are provided as practical guidance and are not product specifications.
A. Labeling an azide-modified protein
Prepare stock: Dissolve the dye in anhydrous DMSO at 5–10 mM. Protect from light.
Buffer: 50 mM HEPES, 150 mM NaCl, pH 7.4 (no sodium azide). Protein at 10–50 µM.
Reaction: Add 2–3 equiv dye per azide site. Incubate 1–2 h at RT with gentle mixing.
Quench/purify: Remove excess dye via spin filtration (30 kDa MWCO) or SEC. Exchange into storage buffer.
QC: Measure A280/A495 and fluorescence (Ex 495/Em 520 nm). Optionally confirm by MS or SDS‑PAGE in‑gel fluorescence.
B. Labeling azide-terminated polymers/nanoparticles
Dissolve substrate in suitable medium (buffer/ACN/DMSO mix). Add 1.5–5 equiv dye.
Incubate at RT for 2–6 h. Purify by precipitation, dialysis, or HPLC as appropriate.
C. Live-cell compatible labeling of azide-labeled glycans (ex vivo)
Wash cells to remove sodium azide/serum. Incubate with dye (1–10 µM final; ≤1% DMSO) in PBS/HEPES for 15–60 min at 37°C protected from light.
Wash thoroughly to remove unbound dye. Analyze by flow cytometry or microscopy using FITC settings.
Notes
Optimize equivalents and time for sterically shielded sites. Maintain low light exposure throughout. Validate cell compatibility for your model system.
Biological Roles
Item-specific biological roles: Not applicable—this is a synthetic, research-use labeling reagent.
General context (literature-based):
Fluorescein is a widely used reporter dye in biochemistry and cell biology due to its high brightness and compatibility with common fluorescence instrumentation (Ex ~495 nm/Em ~520 nm). Signal is pH dependent, with decreased fluorescence in acidic environments (lactone formation).
DBCO enables bioorthogonal conjugation to azide-labeled biomolecules (via SPAAC) without enzymatic pathways or metal catalysts, offering minimal perturbation to biological systems.
dPEG12 linkers can reduce nonspecific binding, improve aqueous solubility, and increase the effective distance between the dye and the biomolecule, which may reduce quenching and improve accessibility in crowded biological environments.
Typical research applications:
Tracking of azide-tagged metabolites (e.g., glycan metabolic labeling) in cells or lysates.
Fluorescent readouts in protein engineering, ligand display, and polymer/material–biomolecule interfaces.
Note: No clinical or therapeutic claims are made. This product is “For research use only.” Users should validate performance in their particular biological system, accounting for fluorescein’s pH sensitivity, potential photobleaching, and cellular compartmentalization effects.
Buffer Applications
Applicability: As a SPAAC labeling probe, buffer choice influences reaction rate, solubility, and fluorescence performance rather than buffering capacity per se.
General buffer guidance (literature/practice):
Suitable buffers: PBS (pH 7.2–7.6), HEPES (pH 7.0–8.0), Tris (pH 7.5–8.0). Maintain near-neutral to slightly basic pH to favor bright fluorescein emission.
Avoid components that complicate downstream analysis:
High concentrations of primary amines are acceptable for SPAAC (since DBCO targets azides), but they can influence protein stability and ionic strength.
Remove sodium azide preservatives prior to labeling of organic azide targets; while inorganic azide does not undergo SPAAC, it can affect certain assays and safety handling.
Ionic strength: Moderate salt (e.g., 150 mM NaCl) typically improves biomolecule stability without hindering SPAAC.
Additives: 0.01–0.05% nonionic surfactants (Tween‑20, Triton X‑100 alternatives) may reduce nonspecific surface adsorption. Reducing agents (DTT, TCEP) are generally compatible with SPAAC but assess impact on your biomolecule.
Practical recipes (example, not specifications):
Labeling buffer: 50 mM HEPES, 150 mM NaCl, pH 7.4; final DMSO ≤10% v/v.
For acid-sensitive fluorescence readouts, ensure final pH ≥7.0 post-mixing; verify with a pH electrode after adding DMSO stocks.
Post-reaction handling:
Purify via SEC/spin filtration in the same buffer to maintain biomolecule integrity and minimize dye hydrolysis/aggregation.
Green Alternatives
Sustainability considerations (general):
SPAAC avoids copper catalysts required for CuAAC, reducing metal waste and simplifying workup—beneficial for green chemistry in bioconjugations.
Use of high‑boiling polar aprotic solvents (DMSO/DMF) has EHS considerations; minimize volumes and prefer aqueous media whenever feasible.
Options and trade-offs (informational; not product specifications):
Solvent choices:
Prefer buffered water with ≤10% DMSO where possible to cut solvent burden.
For synthetic workflows, consider propylene carbonate or ethylene carbonate as greener polar aprotics when compatible, though solubility of large dyes may differ.
Alternative click handles:
Tetrazines (TCO–tetrazine IEDDA) often proceed even faster in water but can suffer from tetrazine instability and potential off-target reactions; DBCO–azide SPAAC is more chemoselective and robust.
2-MeTHF or CPME as workup/extraction solvents can reduce reliance on chlorinated solvents during purification of small-molecule conjugates.
CuAAC (alkyne–azide + Cu): very fast and reliable but introduces copper, necessitating scavenging and posing bio-compatibility concerns.
IEDDA (TCO–tetrazine): ultrafast, no metal; may require careful handling of reactive components and can be costlier.
Best practices:
Scale reactions at concentrations that minimize solvent volume.
Use aqueous purification (SEC, dialysis) to reduce organic solvent use.
Pharmaceutical Uses
This product is not an API or excipient. It is supplied strictly for research use only.
Relevant non-clinical, preclinical, and process development contexts (general):
Conjugation studies in bioconjugate development (e.g., early-stage antibody/PEG/polymer conjugation screening using azide-functionalized candidates) to assess conjugation efficiency by fluorescence.
Analytical tracer in formulation/process characterization—tracking azide-bearing components during purification, filtration, or controlled-release studies at lab scale.
Surface and device prototyping: fluorescent tagging of azide-modified coatings or nanoparticles to visualize coverage and uniformity.
Regulatory notes:
Not manufactured to GMP for human use. No pharmacopeial monograph applies to this specific conjugate.
For any work that might progress toward regulated environments, users should qualify identity and purity via orthogonal methods (MS, HPLC, spectrophotometry) and control residual solvents and endotoxin according to internal SOPs.
Formulation considerations (general):
Prepare stock solutions in DMSO under low light; filter (0.22 µm) if required for sterility-sensitive assays. Use amber vials/syringes to mitigate photobleaching.
Validate stability in intended buffer and at intended storage temperatures (e.g., accelerated and real‑time studies) before adopting into QC or development workflows.
Physical Properties
Item-specific specifications (this SKU):
Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
Appearance: Not specified for this item; refer to CoA/Spec Sheet.
Melting point, boiling point, density, refractive index, UV cutoff, water/peroxide/metal limits: Not specified for this item; refer to CoA/Spec Sheet.
Literature/general properties of the components (for context only; not item specifications):
Fluorescein photophysics (literature):
Absorption maximum ~493–496 nm; emission maximum ~516–520 nm in aqueous buffer near neutral pH.
Quantum yield can approach 0.9 in basic aqueous media; decreases at low pH due to lactone formation.
pKa values for fluorescein acid-base equilibria approximately 2–4.5 and ~6.4 (multiple equilibria; environment-dependent).
dPEG linkers generally increase aqueous solubility and reduce aggregation; actual solubility of this conjugate depends on counterions and formulation (not specified for this item).
Solubility/use guidance (general, not specifications):
Frequently supplied for dissolution in anhydrous DMSO to prepare concentrated stocks (e.g., 1–10 mM), then diluted into aqueous buffers for conjugations.
Fluorescein absorbance/emission are pH sensitive; spectral properties should be recorded under the intended buffer conditions.
Note: For authoritative, lot-specific physical data, consult the CoA/Specification Sheet for SKU D487844.
Quality and Grades
Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
General notes for fluorescent, PEGylated click reagents:
Research grade materials are manufactured for laboratory R&D use and are not intended for use in humans or clinical diagnostics. This product is labeled “For research use only.”
Typical quality attributes for such probes (informational; not specifications):
Identity confirmation by high-resolution MS and/or NMR.
Purity assessment by HPLC/UPLC with UV/Vis detection at the dye’s λmax; sometimes MS-coupled for peak purity.
Counterion content and residual solvents assessed per internal QC methods.
Stabilizers: No stabilizer is specified for this item. If stabilizers or salts are important for your application, verify via CoA/Spec Sheet.
Implications for experimental work:
For quantitative bioconjugation, verify purity and concentration spectrophotometrically (using fluorescein ε at 493–496 nm from literature) and/or by analytical HPLC.
Batch-to-batch comparability: When scaling sensitive labeling workflows, retain a reference chromatogram and emission/absorbance spectrum for in-house QC.
Documentation: Request the lot-specific CoA and SDS from Aladdin Scientific for precise specifications, recommended analytical methods, and acceptance criteria.
Reaction and Applications
Primary application: Copper-free click conjugation (SPAAC) between the DBCO moiety and organic azides.
Use cases (research):
Site-selective labeling of azide-functionalized biomolecules (proteins, peptides, glycoconjugates, nucleic acids) to introduce a fluorescein tag via a hydrophilic PEG12 spacer, minimizing steric perturbation.
Polymer/material modification: grafting fluorescein labels onto azide-terminated polymers, nanoparticles, hydrogels, or surfaces without copper catalysts.
Live-cell compatible labeling of metabolically azide-labeled glycans or lipids (subject to cell permeability; fluorescein signals are pH dependent and can be quenched in acidic compartments).
Mechanistic notes (literature):
SPAAC forms a stable 1,2,3‑triazole at ambient temperature without Cu(I), driven by ring strain in the cyclooctyne. DBCO shows faster kinetics than unstrained alkynes and good selectivity over biological nucleophiles.
Practical tips:
Prepare a fresh, light-protected DMSO stock. Add to azide-bearing target to 1.2–5.0 equivalents relative to azide sites.
Typical conditions: RT, aqueous buffer (pH 7–8), 30 min to several hours depending on sterics and concentration. Gentle agitation improves mixing.
Remove unreacted probe via size-exclusion, spin filtration, HPLC, or dialysis as appropriate.
Verify labeling by fluorescence spectroscopy (Ex ~495 nm/Em ~520 nm, literature), SDS-PAGE in-gel fluorescence, or LC/MS for small molecules/polymers.
Notes:
Minimize exposure to strong light and elevated temperatures during reactions to limit photobleaching.
Avoid sodium azide in buffers which competes with intended azide substrates only if organic azide reactants are present; inorganic azide does not undergo SPAAC but may interfere with certain assays.
Reaction Conditions
General SPAAC conjugation conditions (literature guidance; not item specifications):
Concentrations: Azide‑bearing target 1–100 µM (biomolecules) or 1–10 mM (small molecules/polymers). Use 1.2–5.0 equivalents of DBCO-dye per azide.
Solvent/buffer: Aqueous buffer pH 7.0–8.0 (PBS, HEPES, Tris), with DMSO or DMF cosolvent as needed (final ≤10% v/v for biological systems).
Temperature: Ambient (20–25°C). Lower temperatures slow the reaction; mild warming (up to ~37°C) can accelerate but monitor photobleaching.
Time: 0.5–4 h typical for proteins/peptides; longer for crowded sites or solid supports.
Light: Protect from ambient light; use amber glassware or foil wrap.
Kinetic considerations:
DBCO–azide rate constants are typically in the 10−2 to 1 M−1 s−1 range depending on azide type and medium (literature). Higher concentrations and efficient mixing improve conversions.
Small molecules/polymers: Precipitation, SPE, or HPLC.
Analytical verification:
UV/Vis: Monitor A493–496 nm for fluorescein; correct for baseline.
Fluorescence: Ex ~495 nm/Em ~520 nm; assess labeling degree via calibration curves.
MS: Confirms mass increase corresponding to single or multiple dye additions (depending on azide content).
Compatibility notes:
Reducing agents, salts, and most common buffers are compatible. Avoid competing organic azides in solution other than the target.
Safety and Handling
Regulatory/GHS information for this item:
Signal word, H-statements, GHS classification, pictograms: Not specified for this item; refer to SDS.
General laboratory safety guidance (not a substitute for the SDS):
Handle in a fume hood or well-ventilated area. Avoid inhalation of dust/aerosols and contact with skin/eyes.
Wear appropriate PPE: lab coat, safety glasses, and chemical‑resistant gloves. Use light protection (amber glass/foil) to preserve dye integrity and reduce photobleaching.
Storage incompatibilities (general): Strong oxidizers/reductants may affect the fluorescein chromophore; azide-reactive species are not generally problematic since DBCO is selective for organic azides. Avoid azide salts only if unintended SPAAC with organic azides is a concern.
Hygroscopicity/photolability: Fluorescent dyes can be light sensitive; store protected from light. PEGylated solids/oils may absorb moisture; keep tightly closed with desiccant when possible.
First aid (overview): In case of skin/eye contact, rinse with plenty of water for at least 15 minutes. If inhaled, move to fresh air. If ingested, rinse mouth. Seek medical attention as appropriate.
Spill/cleanup: Absorb with inert material, collect in light-protected container, and dispose according to local regulations.
Always consult the product-specific SDS for authoritative hazard, exposure limits, and disposal instructions.
Solvent Selection
Compound type and polarity (general):
Amphiphilic fluorescent probe: hydrophobic aromatic regions (DBCO, fluorescein) plus a hydrophilic dPEG12 spacer. Overall solubility tends to be enhanced in polar aprotic solvents and mixed aqueous media.
Practical solvent choices (guidance; not specifications):
Stock solutions: Anhydrous DMSO or DMF commonly support high-concentration stocks (e.g., 1–10 mM). Protect from light.
Aqueous buffers: After dilution from DMSO, stable in PBS, HEPES, or Tris buffers near neutral pH for SPAAC labeling. Fluorescein’s photophysics are pH dependent; maintain pH 7.0–8.0 for bright fluorescence.
Miscibility profile (general): DMSO/DMF mix readily with water; gradual addition to minimize local precipitation. The PEG12 segment improves compatibility with water; however, highly saline or very acidic buffers can reduce apparent solubility.
Selection tips:
Choose DMSO when labeling sensitive biomolecules due to its minimal reactivity and high solvating power; limit final DMSO content to ≤10% v/v in biological contexts.
Avoid alcohol-rich media if protein stability is critical. For polymer/material conjugations, acetonitrile or mixed ACN/water (with DMSO cosolvent) can be effective.
Avoid buffers containing sodium azide if labeling azide-modified targets (as a competing reagent). If unavoidable for biostability, remove azide prior to SPAAC via desalting.
Comparison (general):
DMSO: maximal solubilization, biologically tolerated at low %.
DMF: similar solvating power; use for synthetic workflows.
Aqueous PBS/HEPES: biocompatible milieu post-dilution for SPAAC.
Storage and Reconstitution
Item-specific storage and shipping:
Storage conditions: Store at −20°C (per product data). Keep container tightly closed. Protect from light.
General handling and reconstitution (guidance; not specifications):
Upon receipt: Allow to equilibrate to room temperature in the dark before opening to minimize condensation. If provided as a solid, briefly centrifuge to collect material before opening.
Reconstitution: Prepare concentrated stocks in anhydrous DMSO (e.g., 1–10 mM). Vortex gently to dissolve completely. For aqueous work, dilute into buffer with vigorous mixing to avoid local supersaturation/precipitation.
Aliquoting: Divide stock into light-protected, low-dead-volume aliquots to avoid repeated freeze–thaw cycles.
Storage of solutions: DMSO stocks are commonly stored at −20°C protected from light; assess stability periodically by HPLC/UV‑Vis. Avoid >3–4 freeze–thaw cycles.
Stability considerations: Fluorescein can photobleach; minimize light exposure. Avoid prolonged exposure to strong acids/bases and high temperatures.
Documentation:
For any lot-specific guidance on solution stability, counterions, and recommended diluents, consult the CoA/SDS for SKU D487844.
Research use note: For research use only. Not for human or diagnostic use.
Structure and Identity
Item-specific identifiers (CAS, InChIKey, SMILES, formula, exact molecular weight): Not specified for this item; refer to CoA/Spec Sheet.
General structural description (literature):
DBCO-dPEG12-carboxyfluorescein is a bifunctional, modular probe combining:
A DBCO (dibenzocyclooctyne) unit: a strained cyclooctyne fused to two benzene rings; it features a highly reactive internal alkyne for copper-free azide–alkyne cycloaddition (SPAAC).
A dPEG12 linker: a discrete PEG spacer composed of ~12 ethylene glycol repeats providing hydrophilicity, increased aqueous solubility, and spacing to reduce steric hindrance and minimize nonspecific interactions.
A carboxyfluorescein fluorophore: xanthene core with a carboxylate substituent (commonly 5- or 6-carboxyfluorescein). Fluorescein bears phenolic and carboxyl functional groups and exists in pH-dependent ionization states.
Functional groups present (literature): internal alkyne (strained), multiple ethers (PEG), phenols/carboxylate (fluorescein), and aromatic rings.
Stereochemistry: typically none assigned; the architecture is achiral overall unless asymmetry is introduced by substitution pattern on fluorescein.
2D depiction in words: two fused benzene rings curved into a cyclooctyne ring (DBCO) connected via a flexible PEG12 chain to a xanthene-type tricyclic fluorescein bearing a carboxyl substituent.
Intended reactivity handle: The DBCO terminus selectively reacts with organic azides to form stable 1,2,3‑triazoles without copper catalysts (SPAAC).
Synthetic Utility
Role in synthesis and labeling:
DBCO-dPEG12-carboxyfluorescein functions as a ready-to-use, monofunctional labeling reagent that installs a fluorescein tag via the SPAAC reaction onto azide-bearing substrates.
Key functional elements and reactivity (literature):
DBCO strained alkyne: highly selective for organic azides, forming stable 1,2,3‑triazoles under mild, copper-free conditions.
dPEG12 spacer: imparts hydrophilicity and provides a defined separation between the reactive DBCO and the fluorescein reporter, reducing steric clashes and potential fluorescence quenching near surfaces or proteins.
Carboxyfluorescein: robust reporter chromophore; its carboxylate can participate in hydrogen bonding and electrostatic interactions after conjugation. The carboxyl functionality is typically non-reactive under SPAAC conditions but can be leveraged in separate synthetic steps if activated (e.g., NHS ester formation) prior to DBCO installation in custom routes.
Retrosynthetic value:
Enables late‑stage diversification: introduce azide handles onto complex targets (via NHS‑azides, click‑ready sugars, or polymer end‑group azides) and perform a final SPAAC step to append fluorescein without metal catalysts.
Integration with other chemistries:
Orthogonal to thiol–maleimide, SuFEx, and amide couplings, allowing sequential labeling strategies on multifunctional scaffolds.
Compatible with aqueous media, enabling direct conjugation on unprotected biomolecules and water‑swollen materials.
Target Specificity
Item-specific biological target information (antigen, epitope, clone, isotype, species reactivity): Not applicable. This product is a small-molecule fluorescent click reagent and does not possess inherent biological target specificity.
General note:
Specificity arises from the presence of an organic azide on the intended substrate. The DBCO moiety reacts chemoselectively with azides to form a triazole, independent of biomolecular recognition events.
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