Crosslinker Selection Guide

Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents contain reactive ends to specific functional groups (e.g., primary amines or sulfhydryls) on proteins or other molecules.

Choosing among the available options can seem overwhelming until you consider your application needs and categorize these reagents by their general features.

We provide two options for choosing the right crosslinking reagents for your bioconjugation experiments. 

• Try our crosslinker selection tool
• Learn about homobifunctional and heterobifunctional crosslinkers

Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm. Generally, they must be used in one-step reaction procedures to randomly ”fix” or polymerize molecules containing like functional group. This is useful for capturing a snapshot of all protein interactions, but it cannot provide the precision needed for other types of crosslinking applications.

Heterobifunctional crosslinkers possess different reactive groups at either end. These reagents not only allow for single-step conjugation of molecules that have the respective target functional group, but they also allow for sequential (two-steps) conjugations that minimize undesirable polymerization or self-conjugation. In sequential procedures, heteobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. After removing excess unreacted crosslinker, the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs.