Product manual
Product use, preparation, operating conditions and storage.
ATTO dyesReactive dye
ATTO 425 NHS Ester
Cat. No.QH-20-2002
Label proteins, peptides and amino-modified molecules by coupling the NHS ester to accessible primary amines.
Packs & quoteNHS
Activated esterR–NH₂
Primary amineCO–NH–R
Amide linkageExcitation436 nm
Emission484 nm
TargetProtein N-termini, lysine residues and introduced primary amines
Preparation and storage
- Storage
- Store at -20°C, protect from light and moisture
Primary-amine coupling
The NHS ester forms an amide bond with a primary amine; hydrolysis competes with coupling. Proteins usually contain multiple accessible sites, and the input ratio is not the final degree of labeling.
Protein and antibody labeling
For samples with accessible primary amines. Check antigen binding, enzyme activity or the required function after purification.
Amino-modified nucleic acids and peptides
Confirm the number and protection state of amino handles, then choose purification and analysis for the intended product.
Set channels by ATTO catalog number
Check the light source and filters against this product’s excitation and emission values. The number in the product name does not replace the actual wavelengths; recheck negative background and spillover after a channel or conjugate change.
Specifications
Product information
- Product name
- ATTO 425 NHS Ester
- Cat. No.
- QH-20-2002
- Product form
- NHS Ester
- Target
- Protein N-termini, lysine residues and introduced primary amines
- Pack sizes
- 1 mg
Preparation and properties
- Excitation wavelength
- 436 nm
- Emission wavelength
- 484 nm
- CAS
- 892156-28-4
- Molar extinction coefficient
- 45,000 M⁻¹ cm⁻¹
- Quantum yield
- 0.9
- Storage
- Store at -20°C, protect from light and moisture
Spectral information and detection
Product parameters
Use parameters for the exact catalog number when preparing reagents, setting reagent input or calculating results. Request a lot-specific CoA for test results.
Protocol
Buffer
Avoid competing primary amines such as Tris, glycine and free amino acids. Remove carrier proteins such as BSA when they are not intended for labeling. Use a primary-amine-free buffer compatible with the sample.
Dye and input calculation
Prepare the activated ester using a solvent supported for this product. Convert sample and dye amounts to moles using their molecular weights and record the final organic-solvent fraction.
Exchange the sample buffer
Exchange into a suitable buffer and check concentration, purity, pH and visible aggregation.
Set up a reaction series
Use a small sample to compare dye input ratios. Mix gently, protect from light and record pH, temperature and time. Select conditions from this product’s instructions and sample tolerance.
Purification and analysis
Select desalting, size-exclusion separation, dialysis or chromatography for the sample size. Remove free reagent before measuring concentration, labeling density and function. Residual free dye raises background and biases labeling-density calculations.
Assess the conjugate
Calculate labeling density with appropriate extinction coefficients and absorbance corrections, then select conditions based on background, recovery and function.
Request product documentation
Select a document and enter your phone and email. Include the lot number for a CoA.
ATTO 425 NHS EsterQH-20-2002
Spectra and measurement conditions
Spectra, measurement conditions and detection settings for the exact catalog number.
Certificate of analysis (CoA)
Lot-specific test results. Include the lot number from the vial.
Safety data sheet (SDS)
Handling, storage and emergency response information.
Quality and lot information
Unlabeled sample
Use an unlabeled aliquot from the same preparation to assess changes in function, aggregation and recovery.
Free dye and negative sample
Include free-dye or no-target-handle controls to distinguish conjugation from adsorption. Match exposure, gain or voltage during detection.
Input titration
Compare a molar input series and assess signal, negative background, product recovery and function together.
Antibody input and acquisition settings
When comparing ATTO conjugates, use comparable target-protein amounts, labeling-density ranges and acquisition settings, recording specific binding, negative background and recovery.
Technical articles
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.
For protein preparation before NHS-ester labeling; check competing amines and carrier proteins.
Antibody DOL calculation and quality assessment
Calculate DOL with absorbance correction, distinguish input ratio from attached dye, and assess recovery and binding.
For purified protein conjugates; use this dye’s absorbance parameters and distinguish input ratio from DOL.
Application support
Include your sample, formulation and detection method when requesting application guidance.
References
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.
ATTO-TEC primary-amine protein labeling procedures
ATTO-TEC / Leica Microsystems · ATTO Antibody Labeling Kit · 2025-03-19
Manufacturer IgG/NHS-ester example for preparation, purification and absorbance measurements. Its catalog numbers, spectral peaks, extinction coefficients and kit doses do not replace documentation for Qihe catalog numbers.
Frequently asked questions
Can it directly label unmodified nucleic acids?
Routine NHS coupling requires an accessible primary amine. Confirm a suitable amino modification on the oligonucleotide.
Is a higher degree of labeling better?
Overlabeling can affect binding, solubility or signal. Assess specific signal, background and function after purification.
Can aqueous stock be stored for extended periods?
Activated esters hydrolyze in water. Use only the stock solvent, concentration and storage conditions supported for this product.