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Application Protocols
Example protocol A: NHS-first, then maleimide (literature/generic)
Dissolve linker at 10–50 mM in anhydrous DMSO. Keep cold and anhydrous.
Prepare amine-bearing biomolecule in 50–100 mM phosphate or bicarbonate buffer, pH 7.8–8.3 (no primary amines in buffer), 0.15 M NaCl.
Add linker stock dropwise to achieve 1.5–3.0 equiv per accessible amine; keep final DMSO ≤10%.
React 30–60 min at 20–25°C with gentle mixing. Monitor by MS/HPLC.
Quench any residual NHS ester with 20 mM ethanolamine, 10 min.
Desalt (SEC or UF/DF) into maleimide coupling buffer: 50 mM phosphate or HEPES, pH 6.8, 1–5 mM EDTA.
Combine with thiol-bearing partner (or reduce with TCEP just prior). Use 1.1–2.0 equiv maleimide groups per thiol. React 30–90 min at 20–25°C.
Quench excess maleimide with 5 mM cysteine, 10 min. Purify and analyze.
Example protocol B: Maleimide-first, then NHS
Prepare thiol partner in phosphate/HEPES, pH 6.8, with 0.5 mM TCEP (added immediately before use), 1–5 mM EDTA.
Add linker stock (DMSO) to achieve 1.2–2.0 equiv per thiol; react 15–60 min at 20–25°C.
Desalt into pH 7.8–8.3 buffer free of amines. Add amine partner (1–5 equiv to remaining NHS groups).
React 30–90 min; quench with ethanolamine; purify by SEC/UF.
General notes
Verify concentrations by UV or BCA/protein assays as appropriate.
Maintain low oxygen and avoid amine-containing buffers until NHS coupling is complete.
These are example conditions; optimize for your specific system. Consult SDS/CoA for handling constraints.
Biological Roles
Item-specific biological role
This product is a synthetic, heterobifunctional PEG linker for research use only. It is not a natural metabolite or biomolecule.
General context (literature/generic)
Maleimide–NHS PEG linkers are widely used tools in chemical biology for controlled conjugation between thiol- and amine-containing biomolecules. The PEG9 spacer reduces protein–protein and protein–surface nonspecific interactions, improves aqueous solubility of hydrophobic cargos, and can modulate pharmacokinetic properties when incorporated into bioconjugates (conceptual; not a claim for this item).
Maleimide end reacts selectively with cysteine thiols at near-neutral pH, enabling site-directed modification of proteins engineered with unique cysteines. The NHS ester end targets primary amines (lysines, N-termini) under mildly basic conditions.
The orthogonality of these reactions permits stepwise assembly of complex constructs (e.g., fluorophore–protein, protein–polymer, ligand–nanoparticle), facilitating studies in proteomics, imaging, and materials bioconjugation.
Limitations and precautions
Hydrolysis of NHS and ring-opening of maleimide (to maleamic acid) reduce coupling efficiency; timing and pH control are critical.
Competing nucleophiles in buffers (Tris, glycine, ammonia, azide in some contexts) can quench reactivity.
Note
No therapeutic or clinical claims are made. Use is confined to laboratory research by qualified personnel.
Buffer Applications
Applicability
This product is not a classical buffer component. However, successful conjugation depends critically on appropriate buffer choice and preparation (literature/generic guidance below).
Buffers for maleimide–thiol coupling
Composition: 50 mM sodium phosphate or HEPES, pH 6.5–7.0, with 1–5 mM EDTA to suppress thiol oxidation. Optional 0.1 mM TCEP immediately before coupling to maintain free thiols; avoid excess reducing agents that compete with maleimide.
Avoid: Tris, glycine, cysteine, DTT, β-mercaptoethanol, and other nucleophiles.
Ionic strength: 50–150 mM NaCl can improve protein stability without affecting reactivity.
Buffers for NHS–amine coupling
Composition: 50–100 mM phosphate (pH 7.5–8.0) or 50–100 mM bicarbonate/carbonate (pH 8.3–8.5) depending on protein tolerance.
Avoid: Any primary amine buffers (e.g., Tris), hydroxylamine, azide in high concentrations, and high percentages of alcohols.
Operational tips
Degas buffers and filter (0.22 µm) to remove particulates and oxygen; prepare fresh on day of use.
Maintain temperature at 20–25°C to balance kinetics and stability; keep exposure times short to minimize NHS hydrolysis.
Co-solvent: 5–10% anhydrous DMSO can aid solubility upon immediate dilution.
Recipes (examples; scale as needed)
50 mM phosphate, 150 mM NaCl, 1 mM EDTA, pH 6.8 (maleimide step).
100 mM bicarbonate, pH 8.3 (NHS step).
Green Alternatives
Assessment of current practice (literature/generic)
Mal-amido-PEG9-NHS ester generally requires anhydrous, polar aprotic solvents (DMSO/DMF/NMP) for stock solutions to preserve the NHS ester and ensure solubility. These solvents pose environmental and safety concerns, particularly DMF and NMP.
Greener choices and trade-offs
DMSO vs DMF/NMP: DMSO is often the greener choice among polar aprotics due to lower toxicity and better biodegradability. When feasible, prefer DMSO for preparing stock solutions.
Aqueous/biobased co-solvents: Short-term use of buffered aqueous media with 5–20% DMSO can reduce reliance on neat aprotics. Immediate-use strategies limit NHS hydrolysis.
Cyrene (dihydrolevoglucosenone): A bio-based dipolar aprotic solvent with properties similar to DMF/DMAc. Solubility of PEGylated crosslinkers can be favorable; however, water content and reactivity toward NHS esters must be carefully validated at small scale.
Propylene carbonate or ethyl lactate: Sometimes viable for PEG solubilization but may not sufficiently stabilize NHS esters; evaluate compatibility empirically.
Operational green practices
Prepare concentrated stocks to minimize solvent volume.
Use septum-sealed, anhydrous vials to extend shelf life and reduce waste from repeated thawing.
Plan one-pot aqueous dilutions with rapid conjugation to minimize organic solvent use.
Dispose of solvent waste according to local regulations; segregate halogen-free aprotic waste for optimized recycling where available.
Bottom line
Prefer DMSO where possible; explore aqueous immediate-use workflows. Validate any alternative solvent for NHS stability before scale-up.
Pharmaceutical Uses
Item-specific note
No pharmacopeial status or excipient designation is provided for this item. For research use only (per Product Data). Not for human or veterinary use.
Heterobifunctional PEG linkers such as Mal–PEGn–NHS are employed in research and development to assemble bioconjugates (e.g., protein–drug, protein–polymer, ligand–surface) that may be evaluated in pre-formulation and process development studies.
The PEG9 spacer can improve solubility and reduce aggregation during conjugation, facilitating upstream discovery and analytical characterization workflows.
Process considerations
Choice of buffer and order of operations (NHS-first vs maleimide-first) affect conjugation site distribution and product heterogeneity.
Residual linker and hydrolysis byproducts (N-hydroxysuccinimide, maleamic acids) should be controlled by appropriate purification (SEC, ultrafiltration/diafiltration, desalting, HIC) and monitored by LC–MS, UV, and colorimetric assays (e.g., TNBS for free amines, Ellman’s for thiols).
For any movement toward GMP settings, qualified suppliers, comprehensive CoAs, and stability data under ICH conditions are required. This listing does not supply such claims; consult Aladdin Scientific for available documentation.
Stability
NHS esters are moisture sensitive; low-temperature, desiccated storage and anhydrous handling are essential to maintain quality prior to use.
Physical Properties
Item-specific specifications
Appearance: Not specified for this item; refer to CoA/Spec Sheet.
Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
Other specs (water content, residual solvents, UV cutoff, metals): Not specified for this item; refer to CoA/Spec Sheet.
General/literature properties for Mal−PEGn−NHS linkers (informational only; not product specifications)
Physical state: Typically a white to off-white solid for PEG9 chain lengths; hygroscopic and moisture sensitive due to the NHS ester.
Solubility: Readily soluble in anhydrous polar aprotic solvents (DMSO, DMF, NMP, acetonitrile). Soluble to moderately soluble in aqueous buffers when freshly prepared, but NHS ester hydrolyzes in water (literature/generic).
Partitioning: The PEG9 spacer imparts hydrophilicity; overall behavior is amphiphilic, favoring polar media (literature/generic).
Refractive index, logP, pKa: Not typically reported for discrete PEGylated crosslinkers; values depend on exact composition and are not standardized.
Practical implications
Use freshly prepared, anhydrous stock solutions to avoid premature NHS hydrolysis.
In water, maintain near-neutral pH for maleimide-thiol coupling (pH 6.5–7.0) or mildly basic pH for NHS-amine coupling (pH 7.5–8.5). Hydrolysis of the NHS ester accelerates with higher pH and temperature (literature/generic).
Avoid extended exposure to light/heat to minimize maleimide ring hydrolysis to maleamic acid (literature/generic).
Quality and Grades
Item-specific grade/purity
Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
Interpreting grades for PEG crosslinkers (general guidance)
“Bioconjugation” or “crosslinker” grades typically emphasize:
Low residual water (to reduce NHS ester hydrolysis).
Low residual acids/bases and low metal content (to limit side reactions and degradation).
High purity by HPLC (>95–98% for many applications), with narrow distribution of PEG chain length if defined as PEG9 (literature/generic).
HPLC grade solvents (for reconstitution) are preferred to minimize UV-absorbing impurities and moisture.
Stabilizers and implications
These reagents are commonly supplied without stabilizers. Any stabilizer presence should be disclosed on the CoA; none is specified here.
Absence of stabilizers necessitates strict anhydrous handling and cold storage to maximize shelf life.
What to verify on the CoA/Spec Sheet for this item
Assay/purity method (e.g., HPLC, NMR) and acceptance criteria.
Identity confirmation (NMR, MS) for the defined PEG9 length.
Residual solvent and water content (Karl Fischer) if reported.
Recommended storage conditions and retest/expiry date.
Research use note
For research use only (per Product Data). Not for human or veterinary use.
Maleimide with thiols on cysteine-containing biomolecules, peptides, or reduced antibodies.
NHS ester with primary amines (lysines, N-termini) on proteins/peptides or amine-functionalized surfaces/polymers.
Typical applications
Sequential protein–protein or protein–small molecule conjugation.
Site-selective labeling via engineered cysteine residues.
Surface modification of nanoparticles or hydrogels with biomolecules using PEG9 to reduce nonspecific adsorption and improve solubility.
Creation of ADC/bioconjugate mimics and assay reagents in discovery workflows (research use only).
Practical workflow patterns
Two-step coupling: First react NHS end with an amine-bearing partner in slightly basic buffer; purify; then couple the maleimide end to a thiol-bearing partner at near-neutral pH.
Reverse order: First attach maleimide to a thiol-bearing target; then use the NHS end for amine coupling. Choose order based on target stability and the presence of competing nucleophiles.
Key considerations
Maintain anhydrous conditions for NHS activation; control pH stringently (maleimide optimal pH 6.5–7.0; NHS optimal pH 7.5–8.5).
Use reducing agents compatible with maleimide chemistry (TCEP is preferred over DTT/βME which compete as thiols).
Include EDTA (1–5 mM) to prevent thiol oxidation; degas buffers if necessary.
Monitor reactions by MS/HPLC; expect rapid kinetics (minutes to a few hours) depending on concentration and sterics.
Reaction Conditions
General, literature-based guidance (not product specifications)
Stock preparation: Dissolve in anhydrous DMSO or DMF at 10–50 mM. Aliquot and store under inert gas at ≤−20°C. Use within hours to days depending on moisture control.
Maleimide–thiol coupling:
Buffer: 50 mM phosphate or HEPES, pH 6.5–7.0, 1–5 mM EDTA.
Reducing agent: Prefer TCEP (≤0.5–1 mM) immediately before coupling; avoid DTT/βME during reaction.
Temperature: 20–25°C.
Time: 15–120 min typical; monitor by MS/HPLC.
Stoichiometry: 1.1–2.0 equiv linker per thiol for proteins; higher excess for small molecules or sterically hindered sites.
NHS–amine coupling:
Buffer: 50–100 mM phosphate (pH 7.5–8.0) or bicarbonate (pH 8.3–8.5); no primary amines present.
Temperature: 20–25°C.
Time: 15–120 min typical.
Stoichiometry: 1.1–5 equiv depending on target accessibility; consider staged additions to offset hydrolysis.
Order of operations
NHS-first: Useful when amine partner is abundant and thiol partner is sensitive to oxidation.
Maleimide-first: Useful when maintaining thiols in reduced state is straightforward and amine partner is prone to competing side reactions.
Quenching and cleanup
Quench unreacted NHS ester with Tris or ethanolamine after amine coupling (post-reaction only). Quench residual maleimide with cysteine or mercaptoethanol after thiol coupling (post-reaction only). Purify by SEC/UF.
Notes
Oxygen-free, degassed buffers slow thiol oxidation. Maintain low headspace and use freshly prepared solutions to maximize coupling efficiency.
Safety and Handling
Item-specific hazard data
Signal Word: Not specified for this item; refer to SDS.
H-Statements: Not specified for this item; refer to SDS.
GHS Classification: Not specified for this item; refer to SDS.
Pictograms: Not specified for this item; refer to SDS.
General safety information for NHS/maleimide PEG linkers (literature/generic; defer to SDS for authoritative guidance)
Hazards: May cause skin/eye irritation. NHS esters and maleimides are reactive toward nucleophiles (amines, thiols) and can sensitize upon repeated exposure. Handle as a reactive, moisture-sensitive organic solid.
PPE: Use lab coat, nitrile gloves (double-gloving recommended when handling solids/solutions), safety glasses or face shield. Work in a fume hood to avoid inhalation of dust or vapors from organic solvents.
Incompatibilities: Water, alcohols, primary/secondary amines (consume NHS ester), free thiols (consume maleimide), strong bases (accelerate hydrolysis), and strong nucleophiles. Avoid buffers containing Tris, glycine, or other amines during NHS coupling.
First aid (overview):
Skin contact: Wash with soap and water; remove contaminated clothing.
Eye contact: Rinse cautiously with water for several minutes; seek medical attention.
Inhalation: Move to fresh air; seek medical attention if symptoms persist.
Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
Spill/cleanup: Avoid dust formation. Collect solids by gentle sweeping; wipe residues with damp, disposable towels and compatible solvent, disposing as organic hazardous waste.
Stability: Store desiccated at low temperature; minimize exposure to moisture and base to preserve NHS and maleimide functionalities.
Solvent Selection
General solvent behavior (literature/generic)
Polarity: Amphiphilic; the PEG9 chain confers hydrophilicity, while reactive termini favor polar aprotic environments.
Preferred anhydrous solvents for stock solutions: DMSO, DMF, NMP, acetonitrile. These maintain NHS integrity and solubilize PEGylated linkers effectively.
Aqueous use: Short-term in situ dilution into buffered aqueous media is feasible for immediate coupling. NHS hydrolyzes in water; therefore, minimize aqueous residence time.
Buffer compatibility (for conjugation)
Maleimide–thiol coupling: Use phosphate or HEPES at pH 6.5–7.0 with 1–5 mM EDTA to chelate metals that catalyze thiol oxidation. Avoid amines.
NHS–amine coupling: Use phosphate or bicarbonate/carbonate buffers at pH 7.5–8.5. Strictly avoid primary amine buffers (e.g., Tris, glycine) and high concentrations of competing nucleophiles.
Comparison notes
DMSO vs DMF: DMSO is generally less toxic and easier to handle; both dissolve PEG linkers well. DMF has lower viscosity and can facilitate syringe handling for small volumes.
Aqueous co-solvent systems: 5–20% DMSO in buffer can aid solubility while preserving activity when immediate use is planned.
Alcohols (MeOH, EtOH): Can participate as nucleophiles and accelerate hydrolysis; generally avoided for NHS ester stock solutions.
Practical tips
Prepare concentrated anhydrous stocks (e.g., 10–50 mM) and aliquot to minimize freeze–thaw and moisture ingress.
Filter stock solutions (0.2 µm PTFE) if particulates are present; avoid prolonged filtration exposure to moisture.
Storage and Reconstitution
Item-specific conditions (from Product Data)
Storage: Store at −20°C.
Shipping: Ice chest + ice pads.
General handling guidance for maleimide/NHS PEG linkers (literature/generic)
Protect from moisture and light. Store tightly closed in a desiccator or with desiccant in original container at −20°C or below.
Allow the sealed container to equilibrate to room temperature before opening to prevent condensation ingress. Reseal promptly under dry inert gas if possible.
Reconstitution
Solvents: Use anhydrous DMSO or DMF to prepare concentrated stocks (e.g., 10–50 mM). Filter through 0.2 µm PTFE if needed.
Aliquoting: Dispense single-use aliquots into dry, low-bind vials under inert gas to avoid repeated freeze–thaw cycles and moisture exposure.
Aqueous use: Dilute immediately into the appropriate buffer just before conjugation. Keep final organic solvent content ≤10% unless your system tolerates more.
Stability (general, not a specification)
Solid: Stable for extended periods at −20°C when dry and protected from moisture.
Solutions: NHS esters hydrolyze over hours to days depending on water content, pH, and temperature. Prepare fresh solutions for critical work; discard if decreased activity is observed.
Disposal and documentation
Dispose of unused material and solutions as organic hazardous waste according to institutional guidelines.
For exact shelf life, purity, and any stabilizers: Not specified for this item; refer to CoA/Spec Sheet.
Research use
For research use only (per Product Data).
Structure and Identity
Item-specific identifiers (from Product Data)
SKU: M1445534
Product Name: Mal-amido-PEG9-NHS ester
CAS: 2575631-92-2
PubChem CID: 156597084
InChIKey: 194304 (as provided; note that typical InChIKeys are 27 characters; refer to CoA/Spec Sheet for confirmation)
SMILES: Not specified for this item; refer to CoA/Spec Sheet.
Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
Structural features (general/literature description for this linker class)
A thiol-selective maleimide ring (maleimide) at one terminus.
A succinimidyl ester (N-hydroxysuccinimide, NHS) at the other terminus, enabling rapid acylation of primary amines.
A PEG9 spacer (approximately nine ethylene glycol repeat units, –(O–CH2–CH2)9–O–), imparting high aqueous compatibility and reduced nonspecific interactions.
The “amido” designation indicates the maleimide is connected through an amide linkage to the PEG chain, improving stability relative to simple maleimide–alkyl linkers (literature/generic).
2D structure in words (general)
An end-capped linear poly(ethylene glycol) chain of ~9 units links a terminal maleimide ring through an amide bond to a terminal activated carboxyl group converted to its NHS ester. The molecule is overall neutral prior to reaction, with two orthogonal reactive handles: the electron-deficient maleimide double bond and the activated carbonate-like NHS ester. No stereogenic centers are expected in the PEG backbone; the maleimide is planar (literature/generic).
Synthetic Utility
Functional group leverage (literature/generic)
Orthogonal end-groups: maleimide (Michael acceptor for thiols) and NHS ester (activated carboxylate for amines). This permits sequential, chemoselective assembly under mild conditions.
Use cases in synthesis
Protein/peptide modification: Site-selective cysteine labeling via maleimide; lysine/N-terminus acylation via NHS ester.
Materials/surface chemistry: Installation of amine/thiol functionality onto nanoparticles, resins, and hydrogels, using PEG9 to impart antifouling character.
Polymer and small-molecule synthesis: As a modular spacer to couple thiol-bearing and amine-bearing building blocks; facilitates construction of block architectures and multifunctional probes.
Retrosynthetic value
Deconstructs complex bioconjugates into two simpler coupling steps that are tolerant of water, ambient temperature, and physiological ionic strength.
PEG9 segment adds conformational flexibility and distance (~35–40 Å end-to-end when fully extended; literature estimate), minimizing steric hindrance around active sites.
Selectivity and side reactions
Maleimide selectivity for thiols is highest at pH 6.5–7.0; above ~pH 7.5, hydrolysis to maleamic acid and competing amine addition can occur.
NHS esters exhibit rapid aminolysis at pH 7.5–8.5; hydrolysis to the carboxylate competes, especially at higher pH/temperature.
Analytical tracking
Monitor consumption of amines (TNBS/Ninhydrin) and thiols (Ellman’s) to optimize stoichiometry and timing.
Target Specificity
Not applicable to this product type.
This item is a chemical crosslinker, not an antibody, enzyme, or affinity reagent. It does not possess biological target specificity, epitopes, or species reactivity. Any selectivity arises from chemical functionality: maleimide shows chemoselectivity for thiols at near-neutral pH; the NHS ester is reactive toward primary amines under mildly basic conditions (literature/generic).
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