Product manual
Preparation, reaction conditions, purification and storage requirements
AF555 familyAzide click-labeling dye
AF555 Azide
Catalog no. QH-10-1008(Azide)
Pair with a terminal alkyne or a strained alkyne such as DBCO to label a prefunctionalized target with AF555.
Excitation555nm
Emission572nm
Target groupC≡CTerminal / strained alkyne
Reactive formAzide · CuAAC / SPAAC
Spectra and detection conditions ↗Preparation & storage
- Preparation solvent
- Soluble in DMSO or DMF (the labeling reaction is performed in aqueous buffer)
- Storage
- Store at -20°C, protect from light and moisture
AF555 Azide
AF555 Azide supplies an azide handle. Terminal-alkyne targets generally use copper-catalyzed azide–alkyne cycloaddition (CuAAC); strained-alkyne targets such as DBCO use strain-promoted cycloaddition (SPAAC). Both form a triazole linkage but differ in catalyst requirements and sample compatibility.
AF555–N₃
C≡C / DBCO
Triazole linkage
Terminal alkyne: CuAAC. Strained alkyne: SPAAC.
Targets and preparation
| Sample type | Before labeling | Evaluate after labeling |
|---|---|---|
| Terminal-alkyne-modified molecules | Confirm the alkyne and compatibility with copper, ligand and reductant. | Conversion, catalyst cleanup and target function. |
| DBCO-modified proteins or nucleic acids | Confirm available DBCO sites, concentration and prior purification. | Copper-free conjugation, free dye and sample recovery. |
| Alkyne-functionalized surfaces or materials | Distinguish terminal and strained alkynes and include an unmodified-material control. | Signal after washing and nonspecific adsorption. |
Azide does not perform these click reactions directly with ordinary amines or thiols. The sample needs a matching alkyne handle; two azides are not a reactive pair.
Specifications
Product information
- Product name
- AF555 Azide
- Catalog no.
- QH-10-1008(Azide)
- Series
- AF555
- Pack size
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
- Availability and lead time
- Confirmed at the time of inquiry
- Reactive form
- Azide · CuAAC / SPAAC
- Target group
- Terminal / strained alkyne
- Excitation peak
- 555 nm
- Emission peak
- 572 nm
Preparation and properties
- Molar extinction coefficient
- 155,000 M⁻¹ cm⁻¹
- Preparation solvent
- Soluble in DMSO or DMF (the labeling reaction is performed in aqueous buffer)
- Storage
- Store at -20°C, protect from light and moisture
- Molecular weight
- CAS
- 2770422-53-0
- Quantum yield
- 0.1
Optical properties and calculation data
Use this product’s molecular weight and absorbance correction factor for reagent and DOL calculations. Contact technical support for the optical measurement conditions.
AF555 Azide · Protocol
Molar-input calculation
Request the molecular weight for this catalog number before converting mass to molar input.
Before labeling 0 / 4
Identify the reaction pair
Record the alkyne type, available amount and pretreatment. Design CuAAC and SPAAC separately rather than treating all alkyne handles as interchangeable.
Prepare and calculate
Calculate dye moles using this product’s molecular weight. Prepare in DMSO or DMF and control the final solvent fraction. Check clarity and sample stability after mixing.
CuAAC: check the catalyst system
For terminal-alkyne samples, use a validated copper, ligand and reductant system. Assess buffer effects, metal binding and oxidation, with a no-catalyst or no-handle control.
SPAAC: assess concentration and time
Strained-alkyne samples such as DBCO do not require a copper catalyst. Record both reactant concentrations, molar ratio and time, and evaluate conversion and integrity in small-scale trials.
Separate and assess
Remove free dye and, for CuAAC, address residual catalyst components. Choose a separation method for the target’s size and properties, then assess recovery, background and function.
Read the result
- Low recovery
- Check precipitation and purification losses before changing dye input.
- High background
- Separate free dye; compare an unlabeled sample and a target-negative control.
- Reduced function
- Compare pretreatment-only and labeled samples; evaluate lower dye input.
Request product documentation
Choose a document and enter your phone and email. Include the product lot number for a CoA.
AF555 AzideQH-10-1008(Azide)
Certificate of analysis (CoA)
Tests, results and document date for the actual batch
Safety data sheet (SDS)
Hazards, personal protection, handling and disposal information
Supplementary documents
Chemical identity and calculation parameters
Molecular weight, salt form, applicable extinction coefficient and absorbance correction factors
Spectral and application information
Available measurement conditions, spectral records and application notes
Batch quality
Catalog specifications identify the product. Purity, component analysis, test methods and acceptance criteria are supplied in the lot-matched CoA. Include the vial lot number when requesting the report above.
Information for technical support
Include the sample identity, concentration, reactive handles, complete buffer composition, intended application and the question you need to resolve.
Technical articles
Labeling guide
Read guide →CuAAC and SPAAC route selection
Use CuAAC for terminal alkynes and SPAAC for strained alkynes. Identify the handle before assessing copper compatibility.
Labeling guide
Read guide →Troubleshooting low CuAAC conversion
Check reactive handles, catalyst components and copper-binding additives when conversion is low.
Labeling guide
Read guide →Purification and background assessment after click labeling
Use a no-handle control to distinguish labeling from adsorption, and address free dye and catalyst carryover during purification.
Labeling & detection
Read article →AF555 spectra and detection channels
Read the bandpass wavelength range and use single-stain controls to check spillover.
References
References on reaction chemistry, ligand recognition and assay interpretation. Reagents and conditions belong to each study; these papers do not validate Qihe AF product performance or establish equivalence to Alexa Fluor products.
Copper-Catalyzed Azide–Alkyne Click Chemistry for Bioconjugation
Presolski SI, Hong VP, Finn MG. · Current Protocols in Chemical Biology · 3(4):153–162 · 2011
Describes CuAAC catalyst systems and reaction assessment for terminal-alkyne substrates.
Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation
Hong V, Presolski SI, Ma C, Finn MG. · Angewandte Chemie International Edition · 48(52):9879–9883 · 2009
Examines ligand, copper catalyst and biomolecule effects to help troubleshoot low CuAAC conversion.
A Strain-Promoted [3 + 2] Azide–Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems
Agard NJ, Prescher JA, Bertozzi CR. · Journal of the American Chemical Society · 126(46):15046–15047 · 2004
Introduces ring-strain-driven azide–alkyne coupling without copper, a foundational study in SPAAC bioconjugation.
DOI: 10.1021/ja044996f
FAQ
Does AF555 Azide always need copper?
Terminal alkynes generally use copper-catalyzed CuAAC, while strained alkynes such as DBCO use copper-free SPAAC. The partner handle determines the route.
Can it label an unmodified protein?
A matching alkyne handle is required. For ordinary amines or thiols, consider NHS or Maleimide, or introduce an appropriate alkyne first.
Which dye should I use for an azide-bearing sample?
Compare AF555-PEG4-DBCO and AF555-PEG12-DBCO. Two azides do not form this click pair.
Is a copper-free reaction suitable for every live-cell assay?
You still need to assess dye access, handle location, solvent, concentration, background and cell condition. The reaction name alone does not establish suitability.
Will more dye always improve signal?
First distinguish poor conversion from free-dye carryover. More dye can increase purification burden and background; optimize with controls.
Where can I find storage and batch information?
Check the specifications for storage conditions and provide the vial lot number when requesting a CoA. Ask technical support about storage of prepared solutions.
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