Crosslinking reagents homobifunctional

The use of homobifunctional reagents in two-step protocols still creates many of the problems associated with single-step procedures, because the first protein can crosslink and... 

Figure 1.23 Protein crosslinking reactions done using homobifunctional reagents can result in large polymeric complexes of multiple sizes and indefinite structure.

In two-step protocols, one of the proteins to be conjugated is reacted with the homobifunctional reagent and excess crosslinker and by-products are removed. In the second stage, the activated protein is mixed with the other protein or molecule to be conjugated, and the final conjugation process occurs (Figure 4.2). 

The particular crosslinkers discussed in this section are the types most often referred to in the literature or are commercially available. Many other forms of homobifunctional reagents containing almost every conceivable chain length and reactivity can be found mentioned in the scientific literature. 

Figure 4.2 Homobifunctional crosslinkers may be used in a two-step process to conjugate two proteins or other molecules. In the first step, one of the two proteins is reacted with the crosslinker in excess to create an active intermediate. After removal of remaining crosslinker, a second protein is added to effect the final conjugate. Two-step reaction schemes somewhat limit the degree of polymerization obtained when using homobifunctional reagents, but can t entirely prevent it.

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

Bz s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by crosslinking with the amines on both molecules (Figure 22.24). However, single-step crosslinking 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 crosslinker all in solution at the same time. 

Figure 25.10 PEG-amine compounds may be reacted with this heterobifunctional crosslinker to form amide bond derivatives terminating in maleimide groups. This results in a homobifunctional reagent capable of crosslinking thiol molecules. Subsequent reaction with sulfhydryl-containing molecules yields thioether linkages.

Fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinking agents (Chapter 4, Section 4). Their reaction with amines involves nucleophilic displacement of the fluorine atom with the amine derivative, creating a substituted aryl amine bond (Reaction 9). Detection reagents incorporating reactive aryl chemistry include 2,4-dinitrofluorobenzene and trinitrobenzenesulfonate (Eisen et al., 1953). These compounds form... 

The differences within these families of reagents generally relate to the length of the spacer or bridging portion of the molecule. Occasionally, the bridging portion itself is designed to be cleavable by one of a number of methods (Chapter 8). The great majority of homobifunctional, sulfhydryl-reactive crosslinkers mentioned in the literature are not readily available from commercial sources and would have to be synthesized to make use of them. The ones listed in this section are obtainable from Thermo Fisher. 

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