Cross-linking agents are divided into groups dependent on the number and similarity of the reactive groups. Heterobifunctional cross-linkers have two different reactive ends and are used in two step sequential reactions, where the least labile reactive end is reacted first. Heterobifunctional agents allow more controlled two step reactions, which minimises undesirable intramolecular cross-reaction and polymerisation. The most widely used heterobifunctional cross-linking agents are used to couple proteins through amine and sulfhydryl groups. The least stable amine reactive NHS-esters couple first and, after removal of uncoupled reagent, the coupling to the sulfhydryl group proceeds. The sulfhydryl reactive groups are generally maleimides, pyridyl disulfides and α-haloacetyls. Other cross-linkers include carbodiimides, which link between carboxyl groups (-COOH) and primary amines (-NH₂). There are also heterobifunctional cross-linkers with one photoreactive end.
- Reactive toward: Determines the target residues to be cross-linked (select a reagent that does not interfere with protein function)
- Membrane permeability: For cell surface labelling, select non-membrane permeable reagents
- Cleavable: For easy release of cross-linked proteins from solid supports or for further downstream applications
- Reversible: An alternative to cleavable reagents
- Steric hinderance: Bulky groups around the binding site may require reagents with longer spacer arms
Cross-linking agents are used for the following applications: Protein structural and functional studies, cell membrane structural studies, protein and receptor interactions, cell surface studies, solid-phase immobilisation, preparation of immunogens, immunotoxins and other conjugated protein reagents, protein to DNA/RNA cross-linking, reactive group transfer, protein-protein conjugation, and for immunological tools.
To select a cross-linking reagent several factors need to be considered. These factors include:
Ordering information: Double Do™ cross-linkers include the chemical cross-linker, supporting accessories consisting of specific reaction buffers, complete reactions systems, selection guides and protocols.