Crosslinkers Selection Guide

Searching for the perfect crosslinker for your bioconjugation research? Explore our detailed selection guide, conveniently organized by crosslinker type, reactive groups, spacer arm length, and more, to assist you in making the right choice.

Section Overview

• What Are Crosslinkers?
• Crosslinker Selection Reference Table
• Where Can Crosslinkers Be Used?
• Special Considerations When Choosing Crosslinkers

WHAT ARE CROSSLINKERS

In chemical biology and bioconjugation, crosslinkers are essential chemical compounds that connect two or more molecules by forming covalent bonds. These linkers are pivotal in numerous applications, including the creation of bioconjugates, the exploration of protein-protein interactions, and the development of biomaterials. Crosslinkers come in various types, each with specific functionalities tailored to different applications.

Crosslinker Selection Reference Table

Notes:

• DSS (Disuccinimidyl suberate) is a popular homo-bifunctional crosslinker with high reactivity but moderate toxicity.
• Sulfo-SMCC is a widely used hetero-bifunctional crosslinker with high solubility and low toxicity, suitable for in vivo applications.
• EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) is a zero-length crosslinker known for its high efficiency and low toxicity.
• BS3 (Bis[sulfosuccinimidyl] suberate) is similar to DSS but with higher water solubility.
• DTSSP (3,3'-Dithiobis[sulfosuccinimidylpropionate]) offers cleavability under reducing conditions.
• Sulfo-NHS-LC-Biotin is used for biotinylation with a flexible spacer arm.
• BMH (Bismaleimidohexane) provides flexibility with maleimide groups for thiol reactivity.
• DMP (Dimethyl pimelimidate) is less commonly used but effective for specific applications.
• MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester) is another hetero-bifunctional option with specific applications.
• PEGylated Crosslinkers offer customizable spacer lengths and are highly biocompatible.

How Do Crosslinkers Work?

Crosslinkers operate by reacting with specific functional groups on molecules. Amine-reactive groups, such as NHS esters and isocyanates, react with primary amines on proteins or other biomolecules. Sulfhydryl-reactive groups, including maleimides and pyridyl disulfides, react with thiol groups on cysteine residues, while carboxyl-reactive groups, like carbodiimides, activate carboxyl groups to form amide bonds with amines.

WHERE CAN CROSSLINKERS BE USED?

The applications of crosslinkers are broad and varied. They are instrumental in protein-protein interaction studies by stabilizing transient interactions between proteins, which can then be analyzed using techniques like mass spectrometry or Western blotting. In bioconjugation, crosslinkers attach drugs, fluorescent labels, or other probes to biomolecules, facilitating therapeutic development and diagnostics. In material science, crosslinkers help create networks in hydrogels and other biomaterials that can mimic biological tissues. Additionally, they enable the immobilization of biomolecules, such as enzymes or antibodies, on solid supports, enhancing stability and reusability in various assays.

Homo-Bifunctional vs. Hetero-Bifunctional Crosslinkers

Homo-bifunctional crosslinkers, which possess identical reactive groups at both ends, are used to link similar molecules, such as connecting two proteins that share the same functional group. An example of this type is disuccinimidyl suberate (DSS), which features two N-hydroxysuccinimide (NHS) ester groups. In contrast, hetero-bifunctional crosslinkers have different reactive groups at each end, making them ideal for linking diverse molecules, like a protein and a small molecule, each reacting with a different functional group. Sulfo-SMCC, with an NHS ester on one end and a maleimide group on the other, exemplifies this type.

SPECIAL CONSIDERATIONS WHEN CHOOSING CROSSLINKERS

Choosing the right crosslinker involves several key considerations. Specificity is crucial, as the crosslinker must target specific functional groups on the molecules of interest. The length of the spacer arm in the crosslinker affects the distance between linked molecules, influencing the function and stability of the conjugate. Water-soluble crosslinkers are preferred for biological applications to ensure compatibility with aqueous environments. Furthermore, some crosslinkers are designed to be cleavable under certain conditions, allowing for the controlled release of linked molecules.