Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), 50 mg - FAQs

The Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (Cat. No. 22322) maleimide group is stable for 64 hours in 0.1 M sodium buffer, pH 7 at 4 degrees C.

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Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

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Sulfo-SMCC is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended (desiccated at -20 degrees C). Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf).

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SMCC is an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane-stabilized spacer arm (8.3 angstroms). Sulfo-SMCC is a similar compound but it has a sulfonate group on the NHS ring which makes it water-soluble whereas SMCC is water-insoluble and needs to be dissolved first in an organic solvent like DMF or DMSO. Subsequent dilution into aqueous reaction buffer is generally possible, and most protein reactants will remain soluble if the final concentration of organic solvent is less than 10%.

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The Thermo Scientific IP Lysis Buffer (Cat. No. 87787) is a mammalian whole cell lysis buffer based on a modified RIPA buffer formulation without SDS. This moderate strength lysis buffer is able to effectively solubilize cellular proteins but does not liberate genomic DNA or disrupt protein complexes like RIPA buffer.

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The cyclohexane ring in Sulfo-SMCC crosslinker is trans.

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Yes, Sulfo-SMCC can be reconstituted in an organic solvent (DMF or DMSO) and added to an aqueous reaction mixture.

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Yes, Sulfo-SMCC is water soluble.

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Yes, Sulfo-SMCC can be added directly to the reaction mixture.

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No, Sulfo-SMCC cannot be cleaved.

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No, the Sulfo-SMCC cross-linker cannot penetrate a cell membrane.

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Yes, Ellman's reagent (Cat. No. 22582) can be utilized to determine if a protein contains free sulfhydryl groups.

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Yes, Trauts Reagent (Cat. No. 26101) will react with primary amine groups to generate a free sulfhydryl group. SATA (Cat No. 26102) will react with primary amine groups to generate a protected free sulfhydryl group that can be released prior to conjugation.

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No, the disulfides must first be reduced to free sulfhydryls utilizing a reducing agent. If the reducing agent contains free -SH (DTT, B-ME etc.) it must first be removed by desalting. Dialysis may not be used as a substitute for desalting, since over time, disulfide linkages will re-form. 10-50 mM EDTA in the reaction buffer can help slow the re-oxidation of sulfhydryls to disulfides.

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1M Tris (or glycine), pH 7.5 can be added to the reaction mixture to a final concentration of approximately 20-50 mM to quench the NHS-Ester, or the product may be desalted to remove un-reacted cross-linker. Freshly prepared cysteine may be used to quench the maleimide chemistry.

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Long-term storage is not recommended since the NHS-ester group is susceptible to hydrolysis. Hydrolysis begins upon reconstitution. Even organic solvents such as DMSO and DMF are hygroscopic, as a result, they tend to absorb water, which in turn also promotes the hydrolysis of the NHS-Ester.

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A gentle stream of nitrogen gas is held over the opening of the vial to flush the oxygen from the vial. The cap is then immediately placed on the vial. If storing under nitrogen, caution should be taken to ensure that the stream of nitrogen is gentle enough, so that the powdered product will not be blown out of the bottle. If nitrogen is not available, weigh the material as quickly as possible and immediately cap. Store desiccated.

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The product ships under ambient temperature conditions. Upon receipt, the product should be stored at -20 degrees C in a dessicator or with a desiccant bag, and the vial should be warmed to room temperature before opening.

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This product has no set expiration date. We monitor the bulk material in such a manner that allows us to warranty the product for one year from the date of shipment. It may still be usable beyond the one year if handled properly. However, using material older than one year is typically not recommended since the material easily hydrolyzes upon exposure to moisture.

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A 10-20 fold molar excess of cross-linker to protein is recommended for protein solutions greater than 5 mg/mL. A 20-100 fold molar excess of cross-linker to protein is recommended for protein solutions less than 5 mg/mL.

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An initial stock concentration of 5-12 mM (2 - 5 mg/mL) is recommended, or the cross-linking reagent may be added directly to the reaction mixture.

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Buffers containing primary amines, primary sulfhydryls, Tris, ethanolamine, glycine, cysteine or lysine should not be used, since they will quench the reaction. Imidazole, glutathione, or sulfhydryl containing reducing reagents are also not recommended as buffer components.

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We recommend using 100 mM sodium phosphate, 0.15 M NaCl, pH 7.2 (PBS). HEPES, bicarbonate/carbonate, and borate buffers may be substituted for PBS. Other buffers may also be substituted provided they do not contain primary amines or primary sulfhydryls. Recommended pH is 7-9. The reaction can be performed over a pH range of 7-9, however as the pH increases, the rate of hydrolysis of the NHS-Ester also increases as well as the likelihood that the maleimide will react with amines. Once the cross-linker is reconstituted, it must be used immediately.

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A homobifunctional cross-linker has the same reactive group on both ends, for example two amine-reactive groups. It is used in a one-step conjugation where the two molecules are mixed prior to the addition of the cross-linker. This leads to less efficient cross-linking because polymers can easily be created. A heterobifunctional cross-linker has two different reactive groups, for example an amine-reactive group on one end and a sulhydryl reactive group on the other. These can be used in two-step conjugations by reacting the fIrst end with the molecule containing its reactive group, and then adding the molecule that is reactive towards the other end. This leads to much more efficient cross-linking because polymerization does not occur.

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