Product manual
Preparation, reaction conditions, purification and storage requirements
AF750 familyThiol-reactive dye
AF750 Maleimide
Catalog no. QH-10-1015(Maleimide)
Label proteins, peptides and thiol-modified molecules through accessible free thiols.
Excitation753nm
Emission782nm
Target group–SHAccessible free thiols
Reactive formMaleimide · Thiol addition
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 Maleimide
AF750 Maleimide attaches AF750 to a target through maleimide addition to a free thiol. Reactive sites typically come from accessible cysteine residues or introduced thiols; sulfur in disulfide bonds is not counted as a free thiol.
AF750–Maleimide
R–SH
Thioether linkage
Reaction connectivity schematic.
Targets and preparation
| Sample type | Before labeling | Evaluate after labeling |
|---|---|---|
| Proteins with accessible cysteine | Check free-thiol content, accessibility and oxidation state. | Labeling density, recovery and binding or enzyme activity. |
| Thiolated antibodies | Record thiolation or reduction and check antibody integrity. | Aggregation, fragmentation and antigen binding. |
| Peptides and thiol-modified nucleic acids | Confirm thiol deprotection and a suitable separation method. | Product composition, residual dye and function. |
Thiol selectivity does not guarantee a single labeling site. Multiple accessible thiols require site design and product analysis to establish the labeling distribution.
Specifications
Product information
- Product name
- AF750 Maleimide
- Catalog no.
- QH-10-1015(Maleimide)
- 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
- Maleimide · Thiol addition
- Target group
- Accessible free thiols
- 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
- Quantum yield
- 0.12
Optical properties and calculation data
Use this product’s molecular weight and absorbance correction factor for reagent and DOL calculations. Contact technical support for the optical measurement conditions.
AF750 Maleimide · Protocol
Molar-input calculation
Request the molecular weight for this catalog number before converting mass to molar input.
Before labeling 0 / 4
Confirm accessible thiols
Record sample concentration, volume and free-thiol information. Assess structural and functional effects before reducing disulfides, then check the treated sample.
Prepare the reaction buffer
Thiol-containing reductants such as DTT and β-mercaptoethanol compete with the target. Assess TCEP carryover separately. General methods often use pH 6.5–7.5; confirm conditions for this product and your sample.
Prepare the dye and calculate input
Prepare in DMSO or DMF as specified. Use this product’s molecular weight to calculate moles, and record the dye-to-thiol ratio and final organic solvent fraction.
Run a small-scale conjugation
Protect from light and record pH, temperature, time and input ratio. Compare a small set of conditions and retain a sample that underwent the same pretreatment without dye.
Purify and evaluate
Select desalting, size-exclusion chromatography or another suitable separation method. Assess free dye, recovery, labeling density, target function and stability in the intended use environment.
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 MaleimideQH-10-1015(Maleimide)
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
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 →Thiol status and reduction pretreatment
Cysteines may be disulfide-bound or protected. Confirm free thiols before deciding on reduction.
Labeling guide
Read guide →Reductant handling before maleimide labeling
DTT and β-mercaptoethanol compete for maleimide; residual TCEP can also interfere.
Labeling guide
Read guide →Assessment of maleimide-labeled conjugates
Measure labeling density after removing free dye, then check function and changes during storage.
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.
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
FAQ
Can it label an untreated antibody?
The antibody needs accessible free thiols. Decide on reduction or thiolation based on its structure and functional requirements. Consider the NHS route when only primary amines are available.
Does cysteine guarantee single-site labeling?
No. Site number, accessibility and oxidation state determine which residues may react. A single site requires appropriate design and verification.
Should I use an alkaline NHS protocol?
Do not transfer it directly. General maleimide methods usually operate near neutral pH; higher pH can increase hydrolysis and side reactions. Confirm the product-specific conditions.
Can TCEP remain in the reaction?
This depends on concentration and the validated method. TCEP–maleimide interactions have been reported; confirm compatibility or use suitable buffer exchange.
What should I check when signal is weak?
Check free thiols, reductant carryover, sample loss and dye removal, followed by the detection channel and working concentration.
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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