Cross-linking homobifunctional reagents

The problems of indeterminate conjugation products are amplified in single-step reaction procedures using homobifunctional reagents (Chapter 4). Single-step procedures involve the addition of all reagents at the same time to the reaction mixture. This technique provides the least control over the cross-linking process and invariably leads... 

Figure 17 Protein cross-linking reactions done using homobifunctional reagents can result in large polymeric complexes of multiple sizes and indefinite structure.

II Disuccinimidyl tartarate (DST) is a homobifunctional NHS ester cross-linking reagent... 

Avoid covalently linking the A and B chains together during the cross-linking process. This can be done by using heterobifunctional cross-linkers that are more controllable in their reactivity than homobifunctional reagents. 

B/s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by cross-linking with the amines on both molecules (Fig. 353). However, single-step cross-linking procedures using homobifunctional reagents are particularly subject to uncontrollable polymerization of protein in solution. Polymerization is possible because the procedure is done with the liposomes, protein, and cross-linker all in solution at the same time. 

Figure 384 A PEG—amine compound, such as the Jeffamine polymers from Texaco, may be reacted with this heterobifunctional cross-linker to form amide bond derivatives terminating in maleimide groups. This results in a homobifunctional reagent capable of cross-linking thiol molecules. Subsequent reaction with sulfhydryl-containing molecules yields thioether linkages.

All cross-linking agents have at least two reactive groups. In homobifunctional reagents the reactive groups are the same, whereas in... 

---