What factors should I consider when selecting crosslinkers?

Factors one should consider when selecting crosslinkers include: 

• Reagent solubility: The solubility of the cross-linking reagent affects its ability to permeate cell membranes and interact with hydrophobic proteins. Thus, one should consider whether a water-soluble or organic solvent-soluble reagent is needed. 
• Nature of reactive groups: Cross-linkers target specific functional groups on proteins, such as primary amines, carboxyls and carbonyls. One should choose a reagent that does not interfere with the function of the target protein, and consider the number of available functional groups on the protein surface. 
• Chemical specificity: Cross-linking reagents can be homobifunctional or heterobifunctional. Homobifunctional reagents have identical reactive groups and are useful for single-step reactions, while heterobifunctional reagents have two different reactive groups and are used in controlled two-step reactions. 
• Reversible or cleavable: Cleavable cross-linkers allow for the easy release of cross-linked proteins, while reversible cross-linkers enable the recovery and discovery of interacting proteins. 
• Reaction conditions: One should also  consider the optimal reaction conditions, including pH, buffer composition, and cross-linker/protein molar ratios, to achieve the desired results. 
• Spacer arm length: The length of the spacer arm between reactive groups determines the distance between conjugated molecules. Short to medium spacer arms are suitable for intramolecular cross-linking, while longer arms are ideal for intermolecular cross-linking. 
• Photoreactive or thermoreactive groups: Some cross-linkers contain photoreactive groups activated by UV light, while others have thermoreactive groups that spontaneously react.