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AAT Bioquest

What are the factors to consider when choosing a crosslinking reagent?

Posted August 31, 2022


Answer

There are many factors to consider when choosing a crosslinking reagent. They are listed in the bulleted points below.

●       Reagent solubility- The solubility of a crosslinking reagent affects its ability to penetrate cells and conjugate hydrophobic proteins inside membranes. The majority of crosslinkers are soluble in organic solvents (e.g., DMSO, DMF) however some applications require the use of water-soluble crosslinkers.

●       Nature of reactive groups- There are four functional groups that can be targeted for crosslinking methods, (primary amines, carboxyls, sulfhydryls, and carbonyls). One or more reactive groups exist for each target functional group, so one must choose a crosslinking agent that does not interfere with the functions of the target protein. One must also consider the number of functional groups on the surface of the protein. When there are several target groups, in principle, one should use a lower crosslinker/protein ratio, and use a higher crosslinker/protein ratio when there is a low amount of target groups. It's important to note that the lower the number of components, the higher the accuracy during analysis.

●       Chemical specificity (homobifunctional or heterobifunctional):

  • Homobifunctional crosslinking reagents- Have identical reactive groups on both ends of the spacer arm. These are mostly used in single-step reactions and are particularly useful in forming intramolecular crosslinks, polymerizing molecules with similar functional groups, and assessing protein interactions. Common homobifunctional crosslinkers include DTME,DSP, DSs, DST, and BMOE. 
  • Heterobifunctional crosslinking reagents- Have two distinct reactive groups on either end and are used in two-step reactions to reduce unwanted polymerization and cross-reaction. Common examples of these crosslinking reagents include sulfo-SMCC, ANBNOS, ABH, and EMCH. 

●       Photoreactive or thermoreactive groups- Heterobifunctional crosslinkers can have a thermoreactive or spontaneous active group on one end and a photoreactive group on the other. These 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.  

●       Length of the spacer arm- Is this distance between conjugated molecules, ranging from <10 A to >30 A. Crosslinkers with short-medium spacer arms are ideal for intramolecular crosslinking, while longer spacer arms are ideal for intermolecular crosslinking. By using different spacer arm lengths, a broader analysis of protein structure can be viewed.  

●       Reversible or cleavable: A cleavable crosslinker allows for the easy release of crosslinked proteins from solid structures and allows the linkage to be broken when necessary (e.g., for more downstream applications). In turn this allows the molecule to return to its previous state. In contrast, a reversible crosslinker allows for the recovery and identification of interacting proteins. 

●       Potential for additional labeling: Labeling refers to any form of crosslinking that attaches a chemical group to a known protein to assist in the detection, identification, and purification of an unknown protein. Then, the label is cleaved, leaving it attached to the unknown protein.  

●       Reaction condition needed for conjugation: Crosslinking is often performed under mild pH and buffer conditions to maintain the original structure of the protein complex. Optimal crosslinker/protein molar ratios for reactions, the number of functional groups on the protein’s surface, and the degree of conjugation must also be calculated to obtain the expected results.  

Additional resources

Quantitative evaluation of the lengths of homobifunctional protein cross-linking reagents used as molecular rulers

Buccutite™ Rapid Protein Crosslinking Kit

SMCC Plus™ *Enhanced water solubility, crosslinking efficiency and stability*

Buccutite Crosslinkers and Kits