Product manual
Product use, preparation, operating conditions and storage.
Cy3.5Reactive dye
Cy3.5 Maleimide
Cat. No.QH-30-3002(Maleimide)
Label proteins and peptides with accessible cysteine, or thiol-modified molecules, through maleimide–thiol coupling.
Packs & quoteMaleimide
Thiol-reactive endR–SH
Free thiolC–S–R
Thioether linkageExcitation591 nm
Emission604 nm
TargetAccessible free thiols
Thiol addition
Maleimide adds to a free thiol to form a thioether-containing product. Sulfur in disulfide bonds is not an available free thiol; reduction must preserve the required sample structure and function.
Cysteine labeling
For proteins and peptides with exposed free cysteine residues. Site availability depends on the actual thiol state.
Thiol-modified samples
For thiolated antibodies, nucleic acids or materials, with integrity assessed after pretreatment.
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 Maleimide
- Cat. No.
- QH-30-3002(Maleimide)
- Product form
- Maleimide
- Target
- Accessible free thiols
- Pack sizes
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
Preparation and properties
- Excitation wavelength
- 591 nm
- Emission wavelength
- 604 nm
- CAS
- 3077081-59-2
- Molar extinction coefficient
- 116,000 M⁻¹ cm⁻¹
- Quantum yield
- 0.35
- 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
Thiol state
Determine whether thiols are oxidized, protected or buried. If reduction is needed, assess fragmentation, aggregation and function on a small sample.
Reductants and buffer
DTT and β-mercaptoethanol consume maleimide; residual TCEP can also interfere. Remove reductants by a validated procedure and label promptly.
Prepare accessible thiols
Measure or assess free thiols and transfer the sample into a suitable buffer without competing thiols.
Couple protected from light
Set a dye series based on thiol molarity. Record pH, temperature and time, and check for precipitation or aggregation.
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.
Check storage stability
Compare signal, background and function before and after storage. Free-thiol environments can affect the stability of some adducts.
Request product documentation
Select a document and enter your phone and email. Include the lot number for a CoA.
Cy3.5 MaleimideQH-30-3002(Maleimide)
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
Thiol status and reduction pretreatment
Cysteines may be disulfide-bound or protected. Confirm free thiols before deciding on reduction.
For maleimide reactive dyes; first establish accessible thiols on the sample.
Reductant handling before maleimide labeling
DTT and β-mercaptoethanol compete for maleimide; residual TCEP can also interfere.
For maleimide routes; assess reductant carryover and removal.
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
Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with maleimide and alpha-haloacyl groups: anomalous elution of TCEP by gel filtration
Shafer DE, Inman JK, Lees A. · Analytical Biochemistry · 282(1):161–164 · 2000
Examines TCEP interactions with reactive groups, informing evaluation of reductant carryover and buffer exchange.
Tunable degradation of maleimide-thiol adducts in reducing environments
Baldwin AD, Kiick KL. · Bioconjugate Chemistry · 22(10):1946–1953 · 2011
Studies exchange reactions of model adducts in thiol-containing environments to inform conjugate stability assessment.
DOI: 10.1021/bc200148v
Frequently asked questions
Does the presence of cysteine ensure labeling?
No. Disulfides, protecting groups and buried sites can prevent reaction. Confirm accessible free thiols.
Can DTT remain in the reaction?
DTT competes for maleimide and should be removed before labeling using a suitable method.
Must the whole antibody be reduced?
Reduction depends on the intended sites. Excess reduction can damage antibody integrity; assess free-thiol content together with function.
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.