Disuccinimidyl Suberate (Synonyms: DSS, NSC 340008)

The following protocol is an example application for this product. Specific applications will require optimization.
Procedure for Crosslinking Proteins
A. Materials Required
1. Dry dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF) for use with Disuccinimidyl Suberate.
2. Conjugation Buffer: Use a non-amine-containing buffer at pH 7 to 9, such as 100mM sodium phosphate, 0.15M NaCl; 20mM HEPES; 100mM carbonate/bicarbonate; or 50mM borate.
3. Quenching Buffer: 1M Tris•HCl, pH 7.5 (1M glycine or lysine also may be used) − non-reacted reagent can be removed by dialysis or gel filtration.
B. Procedure
1. Prepare protein in Conjugation Buffer.
2. Prepare the crosslinker before use. Dissolve Disuccinimidyl Suberate in DMSO or DMF. Weigh 2mg of Disuccinimidyl Suberate crosslinker into a microcentrifuge tube and add 216μL of solvent to a final concentration of 25mM.
3. Add crosslinker to the protein sample. If the protein concentration is greater than 5mg/mL, use a 10-fold molar excess of the crosslinker. For samples < 5mg/mL, use a 20- to 50-fold molar excess of the crosslinker. Use a final concentration of crosslinker at 0.25-5mM.
4. Incubate the reaction mixture at room temperature for 30 minutes or on ice for 2 hours.
5. Quench the reaction using by adding Quenching Buffer to a final concentration of 20-50mM Tris. Alternatively, remove the non-reacted reagent by dialysis or desalting.
6. Incubate the quenching reaction at room temperature for 15 minutes.

II Intracellular and extracellular cross-linking reactions.

Crosslinking may be performed on cells in suspension or on adherent cells in culture plates. In the latter situation, diffusion of the crosslinking reagent to all surfaces of the cells will be limited and will occur predominately on the exposed surface.Culture media must be washed from the cells otherwise amine-containing components will quench the reaction. Using a more concentrated cell suspension is most effective as less reagent will be required in the reaction. Generally, a final concentration of 1-5mM reagent is effective. NHS reactions occur more rapidly with increasing pH; therefore, pH 8.0 is used in the following example so the reaction can be completed quickly.
A. Materials Required
1.Crosslinker Solution: dissolved the Disuccinimidyl Suberate in dry DMSO at 10-25mM before use.
2.Phosphate-buffered saline (PBS): 20mM sodium phosphate, 0.15M NaCl; pH 8. HEPES, bicarbonate/carbonate or borate buffers between pH 7 and 9 may be used as alternative buffers.
3.Quench Solution: 1M Tris, pH 7.5 (Tris or glycine will quench the reaction.)
B. Procedure
1. Suspend cells at 25×10⁶ cells/mL in PBS (pH 8.0).
2. Wash cells three times with ice-cold PBS (pH 8.0) to remove amine-containing culture media and proteins from the cells. For cell-surface interaction studies, add ligands to the cells and incubate for 1 hour at 4°C.
3. Add the Disuccinimidyl Suberate solution to a final concentration of 1-5mM.
4. Incubate the reaction mixture for 30 minutes at room temperature.
5. Add the Quench Solution to a final concentration of 10-20mM Tris.
6. Incubate the quenching reaction for 15 minutes at room temperature.
NOTE:
1. Disuccinimidyl Suberate is moisture-sensitive. To avoid moisture condensation onto the product, vial must be equilibrated to room temperature before opening.
2. The NHS-ester moiety readily hydrolyzes and becomes non-reactive;therefore, do not prepare stock solutions for storage.
3. Crosslinking proteins with biological activity (i.e. enzymes, antibodies etc.) can result in activity loss upon conjugation possibly caused by conformational changes of the molecule. Activity loss also may occur when the crosslinker modifies lysine groups involved in binding substrate or antigen. Adjusting the molar ratios of reagent to the target may overcome activity loss. Alternatively, use a crosslinker that targets a different functional group.
4. Hydrolysis of the NHS ester is a competing reaction and increases with increasing pH. Hydrolysis occurs more readily indilute protein or peptide solutions. In concentrated protein solutions, the acylation reaction is favored.
References:
[1].Partis M D, Griffiths D G, Roberts G C, et al. Cross-linking of protein by ω-maleimido alkanoyl N-hydroxysuccinimido esters[J]. Journal of Protein Chemistry, 1983, 2: 263-277.