Product manual
Product use, preparation, operating conditions and storage.
Cy3.5Reactive dye
Cy3.5 NHS Ester
Cat. No.QH-30-3002
A Cy NHS label for proteins, peptides and amino-modified molecules with accessible primary amines. Begin protein labeling with a fresh anhydrous stock and a small-scale input series.
Packs & quoteNHS
Activated esterR–NH₂
Primary amineCO–NH–R
Amide linkageExcitation591 nm
Emission604 nm
TargetProtein N-termini, lysine residues and introduced primary amines
Preparation and storage
- Preparation
- Prepare a fresh stock in anhydrous DMSO or DMF; the reference concentration is 10 mg/mL and may be lowered for accurate pipetting. Use promptly after aqueous dilution for NHS labeling.
- Storage
- Store at −20°C, protected from light and moisture. Warm to room temperature before opening to limit condensation.
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.
Multicolor channels and near-infrared detection
Check the light source, filters and detector response against this catalog number’s wavelengths. For near-infrared forms, check detector sensitivity in that range; use separate single-color controls for adjacent Cy channels.
Specifications
Product information
- Product name
- Cy3.5 NHS Ester
- Cat. No.
- QH-30-3002
- Product form
- NHS Ester
- Target
- Protein N-termini, lysine residues and introduced primary amines
- Pack sizes
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
Preparation and properties
- Excitation wavelength
- 591 nm
- Emission wavelength
- 604 nm
- CAS
- 2231670-85-0
- Molar extinction coefficient
- 116,000 M⁻¹ cm⁻¹
- Quantum yield
- 0.35
- Preparation and sample
- Prepare a fresh stock in anhydrous DMSO or DMF; the reference concentration is 10 mg/mL and may be lowered for accurate pipetting. Use promptly after aqueous dilution for NHS labeling.
- Storage
- Store at −20°C, protected from light and moisture. Warm to room temperature before opening to limit condensation.
- Reference pH for NHS protein labeling
- About 8.5
- Reference dye:protein input ratio
- 2–5:1 molar
- Reference reaction time
- 3–5 hours at room temperature
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.
Cy NHS stock
The reference stock is 10 mg/mL in anhydrous DMSO or DMF; reduce the concentration when needed for accurate pipetting. Warm the vial before opening, use reconstituted dye promptly and record organic-solvent fraction and sample clarity in the aqueous reaction.
Prepare protein and buffer
The reference pH for protein labeling is about 8.5 in a compatible primary-amine-free buffer. Remove Tris, glycine and unwanted carrier proteins first, then check sample concentration and integrity.
Set up a Cy NHS input series
For a small protein-labeling trial, use a reference dye:protein molar input of 2–5:1 and react protected from light for 3–5 hours at room temperature. This is an input range, not a target DOL; optimize for sample tolerance, background and binding.
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.
Calculate and compare labeling density
Measure dye and protein molar concentrations after purification, using the extinction coefficient for the exact catalog number and appropriate absorbance corrections. Compare signal, background, aggregation and function at equal protein amounts; total fluorescence is not DOL.
Request product documentation
Select a document and enter your phone and email. Include the lot number for a CoA.
Cy3.5 NHS EsterQH-30-3002
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.
Concentration and self-quenching
Excessive dye loading can cause self-quenching in protein conjugates. Compare purified conjugates made at lower inputs using equal protein amounts and common acquisition settings, assessing signal, background and binding rather than compensating for weak signal by adding more dye.
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
Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin
Gruber HJ, Hahn CD, Kada G, et al. · Bioconjugate Chemistry · 11(5):696–704 · 2000
Compares fluorescence changes when Cy3, Cy3.5, Cy5 and Cy7 associate with IgG or avidin, informing joint assessment of labeling density and the purified conjugate’s signal.
DOI: 10.1021/bc000015m
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.
Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters
Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS. · Bioconjugate Chemistry · 4(2):105–111 · 1993
Studies sulfoindocyanine active-ester structures, spectra and labeling of amine-bearing biomolecules, providing background for cyanine NHS-labeling workflows.
DOI: 10.1021/bc00020a001
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.
Can Cy reactive forms share one parameter set?
Check wavelengths, solvent, molecular weight and correction data by catalog number. Reactive forms sharing a dye name can differ; retain the exact catalog number during selection and comparison.