b'Overview ApplicationsBioconjugation is the process of chemically joining two or moreBioconjugation and crosslinking reagents have a variety of molecules by a covalent bond where at least one molecule isapplications in life science research and assay development. a biomolecule. This technique utilizes a variety of reagents thatThese include fluorescent labeling of proteins and peptides, contain reactive ends to specific functional groups (primaryprotein immobilization onto solid supports, proteinprotein amines, sulfhydryls, etc.) on proteins or other biomolecules. Theconjugation, creation of immunotoxins, and crosslinking for availability of several chemical groups in proteins and peptidesprotein structure and interaction studies. Protein conjugates make them targets for a wide range of applications includingare often designed to enable purification and detection in biotinylation, fluorescence dye conjugation, immobilization tocomplex biological samples. Commonly, antibodies are the solid supports, protein structural studies using crosslinkers, andtarget of bioconjugation with common labels such as biotin or others. Chemical agents may be used to modify amino acidchemically reactive fluorescent dyes. In this process, NHS ester side chains on proteins and peptides in order to alter charges,chemistry is the most widely used method for labeling available block or expose reactive binding sites, inactivate functions, orlysine residues.change functional groups to create targets for crosslinking and labeling. Bioconjugation reagents, crosslinkers, and modificationKey considerationsreagents can be described by their chemical reactivity, molecularBioconjugation and crosslinking reagents are selected based properties, or applications. on their chemical reactivities and other chemical properties that affect their behavior in different applications. Key considerations Chemical reactivity ofinclude the chemical specificity of the reactive ends, reaction bioconjugation and crosslinkingconditions, and whether further modification to the protein or reagents peptide of interest is required to enable bioconjugation. Other important factors that influence the functionality, specificity, and The most important property of a bioconjugation reagentsolubility of the bioconjugation reaction include the spacer arm or crosslinker is its reactive chemical group(s). The reactivelength, cleavability, composition, and structure (Table 1).group establishes the method and mechanism for chemical modification, labeling, or crosslinking. Labeling reagents have a reactive moiety at one terminus, such as an NHS ester for amine labeling, and a chemical moiety at the other, such as a fluorescent dye or biotin, for downstream applications. Crosslinkers contain at least two reactive groups, which target common functional groups found in biomolecules such as proteins and nucleic acids. The functional groups that are commonly targeted for bioconjugation include primary amines, sulfhydryl, carbonyl, and carboxyl groups, and carbohydrates. In addition to targeting functional groups found in biomolecules, bioconjugation reagents and crosslinkers can also employ bioorthogonal groups such as azides and alkynes for click chemistry applications or photoreactive groups for nonselective labeling.102 Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology'