Product manual
Preparation, reaction conditions, purification and storage requirements
AF750 familyAmine-reactive dye
AF750 NHS Ester
Catalog no. QH-10-1015
Fluorescent labeling of primary amines on antibodies, proteins, peptides and amine-modified molecules.
Excitation753nm
Emission782nm
Target group–NH₂Accessible primary amines
Reactive formNHS ester · Amide linkage
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
AF750 NHS Ester
AF750 NHS ester reacts with accessible primary amines to form an amide linkage. Common protein sites include lysine side chains and the N-terminus; peptides, nucleic acids and materials require reactive amines. Free amines and carrier proteins in the sample formulation can also consume dye.
AF750–NHS
R–NH₂
Amide linkage
Reaction connectivity schematic.
Targets and preparation
| Sample type | Before labeling | Evaluate after labeling |
|---|---|---|
| Antibodies and proteins | Check concentration, buffer, carrier proteins and preservatives; exchange buffer when needed. | Recovery, labeling density, aggregation and antigen binding or enzyme activity. |
| Peptides and amine-modified nucleic acids | Confirm amine location, protection state and other potentially reactive amines. | Product composition, unreacted substrate, free dye and target function. |
| Aminated particles or materials | Check accessible surface amines, dispersion stability and the wash method. | Post-wash fluorescence, nonspecific adsorption and particle dispersion. |
Primary-amine reactivity does not guarantee single-site labeling. Evaluate target structure and site design when controlled attachment is required.
Specifications
Product information
- Product name
- AF750 NHS Ester
- Catalog no.
- QH-10-1015
- Series
- AF750
- Pack size
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
- Availability and lead time
- Confirmed at the time of inquiry
- Reactive form
- NHS ester · Amide linkage
- Target group
- Accessible primary amines
- Excitation peak
- 753 nm
- Emission peak
- 782 nm
Preparation and properties
- Molar extinction coefficient
- 290,000 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
- 1300 g/mol
- A280 correction factor (CF)
- 0.04
- Suggested IgG DOL
- 2–5
- Reaction buffer
- IgG example: 0.1 M bicarbonate, pH approximately 8.3
- DMSO stock storage
- Anhydrous DMSO stock: aliquot at −20°C protected from light, use within one month and avoid moisture and repeated freeze–thaw
- Quantum yield
- 0.12
Optical properties and calculation data
CF corrects dye absorbance at 280 nm: corrected A280 = A280 − CF × Amax. The suggested DOL is the average number of dyes per IgG, not the input ratio. Confirm binding activity after purification; these parameters apply to this NHS ester.
AF750 NHS Ester · Protocol
Stock-solution calculation
1 mg ÷ 1300 g/mol ÷ 10 mmol/L = 76.9 μL
Calculated solvent volume for 1 mg at 10 mM. Actual working concentration must remain within the product’s solubility limits.
- Solvent
- Anhydrous DMSO or DMF
- Buffer
- IgG example: 0.1 M bicarbonate, pH ≈ 8.3
- Reaction example
- IgG: 1 h at room temperature, protected from light.
- DMSO stock
- Aliquot, protect from light and store at −20°C for up to one month. Avoid moisture and repeated freeze–thaw.
Before labeling 0 / 4
Review the formulation and exchange buffer
Primary-amine components such as Tris and glycine compete with the target; carrier proteins such as BSA may also be labeled. Determine whether they need removal, then record sample concentration and recovery after buffer exchange.
Set pH and a small-scale comparison
The AF NHS manual uses bicarbonate buffer at approximately pH 8.3 in its IgG example. Confirm sample stability under these conditions; increasing pH also accelerates NHS ester hydrolysis.
Prepare fresh dye and calculate molar input
Prepare in DMSO or DMF as specified, limit moisture exposure and use promptly. Calculate dye moles using this product’s molecular weight and record the final organic solvent fraction. Equal masses of different dyes do not represent equal molar inputs.
Conjugate under light protection
Record sample concentration, dye-to-target molar ratio, pH, temperature and reaction time. Compare a limited input series and retain an identically handled sample without dye for functional comparison.
Remove free dye and evaluate
For proteins, select desalting or size exclusion according to size. Small molecules and peptides require a method that resolves substrate, product and dye. After purification, check recovery, residual dye, labeling density 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.
AF750 NHS EsterQH-10-1015
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
AF NHS parameters and preparation
Molecular weight, extinction coefficient, A280 correction factor and suggested IgG DOL
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 →Buffer checks before NHS labeling
Tris and glycine consume NHS dye. BSA and gelatin require separation from the target protein; routine buffer exchange is insufficient.
Labeling guide
Read guide →Troubleshooting low NHS labeling signal
Check NHS hydrolysis and amine-containing additives, then distinguish low labeling from sample loss or reduced binding.
Labeling guide
Read guide →Antibody DOL calculation and quality assessment
Calculate DOL with absorbance correction, distinguish input ratio from attached dye, and assess recovery and binding.
Labeling & detection
Read article →AF750 spectra and detection channels
Read the bandpass wavelength range and use single-stain controls to check spillover.
Labeling & detection
Read article →NHS and maleimide labeling routes
NHS labels primary amines; maleimide labels free thiols. Additives and reduction requirements determine sample preparation.
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.
Labeling a protein with fluorophores using NHS ester derivitization
Nanda JS, Lorsch JR. · Methods in Enzymology · 536:87–94 · 2014
Uses FAM ester labeling of a protein to explain primary-amine chemistry and labeling-efficiency assessment. It informs NHS workflow design; input and reaction conditions depend on the actual dye and sample.
Fluorescent-labeled antibodies: Balancing functionality and degree of labeling
Vira S, Mekhedov E, Humphrey G, Blank PS. · Analytical Biochemistry · 402(2):146–150 · 2010
Uses FITC-labeled anti-HA antibody to assess changes in labeling density and antigen binding. It informs input-series design and post-purification functional testing; DOL alone is insufficient for selecting a conjugate.
FAQ
Can an antibody containing Tris or BSA be labeled directly?
Tris competes through its primary amine, and BSA can also be labeled. Review the complete formulation and assess buffer exchange or carrier removal before attributing labeling and signal to the target antibody.
Is higher dye input or labeling density always better?
Assess labeling density together with binding, background and recovery. Excessive labeling may affect target function; determine suitable conditions through a small input series and application testing.
Can NHS ester directly label a nucleic acid without an introduced amine?
This route targets accessible primary amines and generally uses nucleic acids with a defined reactive amine modification. Match the route to the actual handle; alkynes or azides may support the corresponding click-labeling products.
Where can I obtain molecular weight and labeling calculations?
Specifications lists this product’s molecular weight, extinction coefficient, A280 correction factor and suggested IgG DOL. Request full parameter sheets, instructions and lot documents under Documents & quality. Suggested DOL refers to the purified conjugate’s average labeling density, not the reaction input ratio.
Can purification be skipped after labeling?
Residual dye affects absorbance, fluorescence and background assessment. Choose purification appropriate to target size and confirm adequate separation of free dye from the labeled product.
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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