Product manual
Preparation, reaction conditions, purification and storage requirements
AF546 familyCopper-free click-labeling dye
AF546-PEG4-DBCO
Catalog no. QH-10-1007(DBCO PEG4)
Label azide-bearing proteins, nucleic acids or other molecules through a PEG4 spacer and a DBCO handle.
Excitation554nm
Emission570nm
Target group–N₃Azide groups
Reactive formPEG4-DBCO · Copper-free 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
AF546-PEG4-DBCO
AF546-PEG4-DBCO combines AF546, a PEG4 spacer and a DBCO handle. DBCO reacts with target azides by strain-promoted azide–alkyne cycloaddition (SPAAC), forming a triazole linkage without a copper catalyst. The shorter PEG4 spacer can be evaluated alongside PEG12 to assess the effects of linker distance on the sample.
AF546–PEG4-DBCO
R–N₃
SPAAC triazole linkage
Reaction connectivity schematic.
Targets and preparation
| Sample type | Before labeling | Evaluate after labeling |
|---|---|---|
| Azide-modified proteins or antibodies | Confirm azide loading, sites and prior purification. | Recovery, labeling density, aggregation and binding. |
| Azide-modified peptides or nucleic acids | Confirm intact azides and calculate input from the specific molecular weights. | Product composition and separation from unreacted dye. |
| Azide-functionalized surfaces or materials | Assess handle accessibility and dispersion, with a no-azide control. | Post-wash signal, background and material function. |
PEG4 and PEG12 differ in molecular weight and spacer length. Compare at matched molar input and assess solvent requirements, recovery and function separately.
Specifications
Product information
- Product name
- AF546-PEG4-DBCO
- Catalog no.
- QH-10-1007(DBCO PEG4)
- Series
- AF546
- Pack size
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
- Availability and lead time
- Confirmed at the time of inquiry
- Reactive form
- PEG4-DBCO · Copper-free SPAAC
- Target group
- Azide groups
- Excitation peak
- 554 nm
- Emission peak
- 570 nm
Preparation and properties
- Molar extinction coefficient
- 112,000 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
- Quantum yield
- 0.79
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.
AF546-PEG4-DBCO · Protocol
Molar-input calculation
Request the molecular weight for this catalog number before converting mass to molar input.
Before labeling 0 / 4
Confirm the azide-bearing sample
Check azide introduction, available sites and prior purification. Remove free azide species that may consume DBCO; assess sodium-azide-containing storage buffers and exchange them as appropriate.
Prepare at the required molar amount
Calculate input using the molecular weight of AF546-PEG4-DBCO, prepare in DMSO or DMF and record the final organic solvent fraction.
Perform copper-free conjugation
Add the dye in a buffer compatible with the sample without a copper catalyst. Record reactant concentrations, ratio, pH, temperature and time, and protect from light.
Compare concentration and spacer length
Compare a small set of concentrations and times. If accessibility may be limiting, include a parallel PEG12 condition while keeping other variables matched.
Purify and validate the product
Remove free dye using separation or washing suited to the target. Assess labeling with a no-azide control, recovery, labeling density and functional testing.
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.
AF546-PEG4-DBCOQH-10-1007(DBCO PEG4)
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 →PEG4 and PEG12 spacer comparison
PEG4 and PEG12 differ in spacer length. Compare labeling, function and background at matched molar input.
Labeling guide
Read guide →Sample and buffer checks before SPAAC
Target-bound azides enable coupling; free azide reagents and sodium azide can compete for DBCO.
Labeling guide
Read guide →Troubleshooting weak signal and high background after SPAAC
Use product analysis to separate low conversion from detection bias, and a no-azide control to check adsorption.
Labeling & detection
Read article →AF546 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.
Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition
Debets MF, van Berkel SS, Schoffelen S, et al. · Chemical Communications · 46(1):97–99 · 2010
Reports copper-free reaction of a strained alkyne with azides and its application to enzyme PEGylation.
DOI: 10.1039/b917797c
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 the reaction need copper?
DBCO reacts with azides through SPAAC without a copper catalyst. Do not transfer a terminal-alkyne CuAAC catalyst recipe to this reaction.
Does it react directly with protein amines?
The reaction partner is an azide. Introduce azides into an ordinary protein first, or use AF546 NHS for primary-amine labeling.
Can PEG4 replace PEG12 at the same mass?
They have different molecular weights, so equal masses are not equal molar amounts. Obtain both values and recalculate.
What if the sample contains sodium azide?
Assess and remove free azide species as appropriate to avoid competing DBCO consumption. Recheck sample condition and concentration after buffer exchange.
Can I prepare it in water or PBS?
Use DMSO or DMF as documented for this product. Solubility and usable concentration in water require separate confirmation.
Can it be used directly in live cells?
Copper-free means no copper catalyst is needed. Dye access, handle accessibility, solvent, cell condition and background still require validation.
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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