Antibody and protein labeling
Prepare your own AF405 conjugate. Retain sample-lot, input-ratio and purification records for reproducibility.
AF405 familyAmine-reactive dye
AF405 NHS Ester
Catalog no. QH-10-1002
For conjugation to proteins, antibodies and other molecules with accessible primary amines.
Excitation400nm
Emission424nm
Target group–NH₂Accessible amines
Reactive formNHS active ester · Forms an 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
AF405 NHS ester for fluorescent amine labeling
AF405 NHS ester is an amine-reactive dye in Qihe’s AF series. Its NHS active ester reacts with accessible primary amines to form amide linkages. Relevant sites include lysine side chains and available N-terminal amino groups on proteins and antibodies.
The excitation maximum is 400 nm and the emission peak is 424 nm. Prepare and purify the labeled molecule, then assess binding, background and fluorescence for the intended application.
AF405–NHS
R–NH₂
Amide linkage
Reaction connectivity schematic.
Targets for NHS labeling
| Sample type | Before labeling | Evaluate after labeling |
|---|---|---|
| Purified antibody / protein | Accessible primary amines; Tris, glycine, BSA or gelatin in the storage buffer | Residual free dye, degree of labeling and binding or function |
| Peptides with primary amines | Whether amino groups are blocked and whether multiple sites may react | Product composition, separation and target activity |
| Amine-modified oligonucleotides | Available introduced amino groups and purification requirements | Separation of labeled product and suitability for the nucleic-acid application |
Peptides and amine-modified nucleic acids are general targets for NHS chemistry. Conditions for a specific sample with this product require separate confirmation. For a defined single site, plan the site design first; conventional NHS labeling does not guarantee a single labeling position.
Labeling and detection applications
Flow cytometry
Evaluate the conjugate against the laser, filters and other dyes. Include unstained and single-stained controls and retitrate the working amount.
Fluorescence imaging
Check the excitation source and emission collection range. Evaluate signal and background using the actual sample and exposure conditions.
If the sample provides free thiols or azides, compare other AF405 reactive forms first. View related products →
Specifications
Product information
- Product name
- AF405 NHS Ester
- Catalog no.
- QH-10-1002
- Series
- AF405
- 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 active ester
- Target group
- Accessible primary amines
- Excitation peak
- 400 nm
- Emission peak
- 424 nm
Preparation and properties
- Molar extinction coefficient
- 35,000 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
- 1028.3 g/mol
- A280 correction factor (CF)
- 0.7
- Suggested IgG DOL
- 5–10
- 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
- Molecular formula
- C46H69N5O15S3
- Appearance
- Off-white powder
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.
AF405 NHS Ester · Protocol
Stock-solution calculation
1 mg ÷ 1028.3 g/mol ÷ 10 mmol/L = 97.2 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.
AF405 NHS EsterQH-10-1002
Product manual
Preparation, reaction conditions, purification and storage requirements
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
Information for technical support
For purchasing:Catalog number, pack sizes and quantities, required delivery date and experimental application.
For labeling:Sample identity, concentration and available amount, full buffer composition, instrument configuration and questions to resolve.
For a purchased batch:Catalog number, vial lot number and document type. For experimental problems, also provide the actual procedure and control results.
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.
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.
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.
AF405 NHS FAQ
Which groups does AF405 NHS label?
Accessible primary amines, such as protein N-terminal and lysine side-chain amino groups, are the main targets. Site number and accessibility affect the product. Conventional NHS coupling does not guarantee a single labeling site.
Can samples containing Tris or glycine be labeled directly?
These primary-amine components compete in the reaction. Assess buffer exchange and sample stability before confirming the reaction system. Adjusting pH alone does not remove competition.
Is buffer exchange sufficient for antibodies containing BSA or gelatin?
BSA and gelatin are also labelable proteins. Routine dialysis or ultrafiltration buffer exchange cannot reliably separate them from the antibody. Prefer carrier-free samples or a purification method suitable for that sample.
Does this product need EDC?
This product is already an active NHS ester. The relevant reaction is active-ester coupling to primary amines; do not add the EDC / NHS activation step used for carboxyl substrates directly to this workflow. Confirm the specific formulation in the product instructions.
Should I use water, DMSO or DMF to prepare the dye?
Prepare the dye in DMSO or DMF; labeling takes place in aqueous buffer. Confirm the stock concentration and solvent fraction against the product instructions and sample tolerance.
How long can a prepared dye solution be stored?
Dye solutions require different storage conditions from the solid. Contact technical support with the solvent, concentration and preparation date for storage advice.
Does a tenfold molar input mean a DOL of 10?
Input ratio describes added amounts; DOL is the average number of dyes bound per protein after purification. Unreacted dye and purification losses affect the final result. Record the two values separately.
Is brighter signal or higher DOL always better?
Also assess background, binding and working concentration. Excess labeling can affect molecular function, and some dyes can self-quench. Establish suitable conditions using the actual conjugate.
Why is background high after labeling?
Check free-dye removal first, then compare blanks, unlabeled samples and different working concentrations. Retain instrument settings to distinguish sample background from conjugate signal.
Does a 405 nm laser guarantee suitability?
The excitation and emission maxima are 400/424 nm. Check the emission collection window, optics and sample background, then validate with the actual conjugate and controls.
Can I use another brand’s AF405 molecular weight or formulation?
This product’s identity, salt form, molecular weight and validated conditions require independent confirmation. Other brands’ public data can inform background reading but must not be inserted into QH-10-1002 parameters or calculations.
How do I obtain a batch CoA?
Prepare catalog number QH-10-1002 and the lot number on the vial, then request the document. Check its catalog number, lot number, date and applicable version on receipt.