Disuccinimidyl suberate

Disuccinimidyl suberate is a six-carbon lysine-reactive non-cleavable cross-linking agent.
It consists of functional groups It is a homobifunctional N-hydroxysuccinimide ester formed by carbodiimide-activation of carboxylate molecules, with identical reactive groups at either end. The reactive groups are separated by a spacer and in this molecule it is a six carbon alkyl chain. This reagent is mainly used to form intramolecular crosslinks and preparation of polymers from monomers. It is ideal for receptor ligand cross-linking.
DSS is reactive towards amine groups at pH 7.0-9.0. It is membrane permeable, therefore permitting intracellular cross-linking, has high purity, is non-cleavable, and is water-insoluble
Its reaction specificity, reaction product stability, and lack of reaction by-products make it a commonly used cross-linking agent.

Applications

Chemical crosslinking of intracellular proteins prior to cell lysis and immunoprecipitation
'Fix' protein interactions to allow identification of weak or transient protein interactions
Protein crosslinking to create bioconjugates via single-step reactions
Immobilize proteins onto amine-coated surfaces
Crosslinking mass spectrometry provides insight into protein structure, organization, and interactions

Size exclusion chromatography

A study was done looking at the application of size exclusion chromatography in the purification of cross-link peptides within samples. When the eluted peptides were analyzed, cross-linked peptides could be detected at higher concentrations in the earlier fractions, eluting before modified or unmodified peptides.

Cross-linking with hemoglobin-albumin

Disuccinimidyl suberate's reactivity toward primary amines allows it to serve as a cross-linking agent for proteins, without toxic side-products and forming peptide bonds with the lysine residues in a single step. In a study on blood substitutes, DSS was shown to cross-link Hemoglobin intramolecularly, yielding a relatively stable protein, whose oxygen affinity was almost halved compared to that of native Hb. This was shown to be reversed when Hemoglobin was copolymerized with bovine serum albumin, showing very little change in auto-oxidation and oxygen affinity compared to the native Hb.