SMCC (Succinimidyl 4-(N-maleimido-methyl)-cyclohexane-1-carboxylate) and Sulfo-SMCC (Sulfosuccinimidyl 4-(N-maleimido-methyl)-cyclohexane-1-carboxylate) are heterobifunctional crosslinkers commonly used for labeling biomolecules or cross-linking oligonucleotides, peptides, and proteins. Both molecules have two reactive groups, an NHS ester group that reacts with primary amines (-NH₂) on oligonucleotides, peptides, proteins, or other biomolecules, forming a stable amide bond and a maleimide group that reacts with thiol (-SH) groups on other molecules, forming a stable thioether bond.
| SMCC: Succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, C16H18N2O6. Mw 334.33. | Sulfo-SMCC: Sulfosuccinimidyl 4-(N-maleimido-methyl) - cyclohexane-1-carboxylate), C16H17N2O9S. Mw 436.37. |
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| Moisture-sensitive. Store at 4 ºC | Moisture-sensitive. Store at - 20 ºC |
Both molecules are moisture-sesnsitive. Store desiccated and equilibrate vial to room temperature before opening to avoid moisture condensation inside the container.
The spacer arm adds a mass of 219.09 mass units and a linker length of ~ 8.35 Å to the final conjugates. The two-step reaction scheme for conjugating two selected molecules, R1 and R2, is shown as follows.
Step one: Labeling the amino group.
Step 2: Thiol reaction to generate a cross-linked molecule.
SMCC Guidelines for Standard Labeling Process
Activate first molecule with SMCC: Dissolve SMCC in a suitable organic solvent, for example, DMSO or DMF. Add it to a biomolecule containing primary amines. Incubate for 30 to 60 minutes at room temperature (pH 7.4–8.5). Remove excess unreacted SMCC via desalting or dialysis.
React labeled molecule with Thiol-Containing Molecule: If necessary, reduce disulfide bonds to generate free thiols by using TCEP or DTT. Add the thiol-containing molecule to the SMCC-modified biomolecule. Incubate at pH 6.5 to 7.5 for 1 to 2 hours.
Purify: Remove unreacted components using size-exclusion chromatography, dialysis, or ultrafiltration.
Sulfo-SMCC
Sulfo-SMCC is a water-soluble, heterobifunctional crosslinker commonly used for conjugating amine-containing biomolecules such as oligonulceotides, peptides, proteins, and antibodies to thiol-containing molecules including drugs, oligonucleotides, peptides, proteins, and fluorophores. Unlike SMCC, it does not require organic solvents for dissolution.
Sulfo-SMCC Labeling Protocol
Activation of Amine-Containing Compounds: Dissolve the molecule selected for conjugation in a buffer, for example, using PBS (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2), or 50 to 100 mM sodium phosphate, pH 6.5 to 7.5, the addition of 1 to 5 mM EDTA can help to chelate divalent metals. (Use amine- and sulfhydryl-free buffer). Add Sulfo-SMCC (freshly prepared in water) at a molar ratio of ~ 2:1 to up to 10:1 (e.g. crosslinker:protein). Incubate at room temperature for 30 - 90 minutes. Remove excess Sulfo-SMCC via desalting, for example by using spin columns, or dialysis.
Reaction with Thiol-Containing Molecule: If necessary, reduce disulfide bonds to generate free thiols using TCEP or DTT. If using DTT, remove excess DTT before conjugation reaction. Add the thiol-containing molecule to the Sulfo-SMCC-activated compound. Incubate at pH 6.5 - 7.5 for 1 - 2 hours at room temperature.
Purification of Conjugate: Use gel filtration, size-exclusion chromatography, or dialysis to remove unreacted components.
Conjugation Efficiency: Conjugation efficiency for most conjugates can be estimated or even quantified by electrophoresis separation and staining, or, better, buy MALDI-TOF-MS.
Typical applications
Antibody-drug conjugation (ADCs)
Bioconjugation enables attaching peptides, oligonucleotides, or other biomolecules to other selected biomolecules.
Drug conjugation for delivery is useful for linking drugs to carrier oligonucleotiedes, PNAs, peptides, proteins or nanoparticles.
Enzyme conjugation or labeling
Fluorophore conjugation or labeling
Nanoparticle functionalization
Oligonucleotide conjugation or labeling
DNA-peptide conjugation
Oligonucleotide-protein conjugation
Protein conjugation or labeling is useful for conjugating biomolecules such as antibodies, oligonucleotides, peptides, and proteins to fluorophores or enzymes.
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