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AAT Bioquest

What are the different types of crosslinking reagents?

Posted August 31, 2022


Answer

There are three different types of crosslinking reagents, heterobifunctional, homobifunctional, and photoreactive crosslinkers. Each reagent’s characteristics are explained in the bulleted points below.

●       Homobifunctional crosslinking reagents- Have identical reactive groups on both ends of the spacer arm. These reagents are mostly used in single-step reactions and are particularly useful in forming intramolecular crosslinks, polymerizing molecules with similar functional groups, preparing polymers from monomers, and assessing protein interactions (although they don’t have the accuracy required for other types of crosslinking applications). Common sulfhydryl-to-sulfhydryl crosslinkers include DTME and BMOE. Amine-to-amine crosslinkers include DSS, DST (used for applications where crosslink cleavability is required while keeping protein disulfide bonds intact), and DSP (ideally used for crosslinking intracellular proteins prior to cell lysis and immunoprecipitation, also for fixing protein interactions prior to identifying weak or fleeting protein interactions).   

●       Heterobifunctional crosslinking reagents- Have two distinct reactive groups on either end of the spacer arm; these reagents can be used to link heterogenous functional groups. They produce multiple intermolecular crosslinks and conjugates using different molecules. Unlike homobifunctional crosslinkers, they utilize two-step reactions which minimizes unwanted polymerization or self-conjugation. Common examples of these crosslinking reagents include MDS, EMCS, GMBS, sulfo-EMCS, and ABH.

●       Photoreactive or thermoreactive groups- Are heterobifunctional crosslinkers can have a thermoreactive or spontaneous active group on one end and a photoreactive group on the other. They become reactive only upon exposure to UV or visible light. This crosslinker is best used for non-specific conjugation, and can be used to bind proteins, nucleic acids, and other molecular structures. The two most widely used photoreactive chemical groups used in science are aryl-azides and diazirines. 

N-((2-pyridyldithio)ethyl)-4-azidosalicylamide are the most common photoreactive reagents in crosslinking reactions. Upon exposure to 250-350 nm UV light, these crosslinkers help assist in the formation of a nitrene group and initiate the production of hydrocarbon insertion products. ANB-NOS, and Sulfo SANPAH are more examples of aryl azides.   

NHS ester diazirines have a photoactivatable diazirine ring and NHS ester that reacts with primary amino groups to form stable amide bonds. 

Additional resources

Chemical cross-linking in the structural analysis of protein assemblies

Buccutite Crosslinkers and Kits

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