Crosslinking is the process of the covalent binding between two or more molecules. The crosslinking technique involving proteins and other biomolecules is often referred to as bioconjugation. Protein crosslinking strategies are carried out by targeting (i) the primary amine including the N-terminal amine in each polypeptide chain and lysine side chain, (ii) the carboxyl group in the C-terminus of each polypeptide chain and in the side chains of the aspartic acid and glutamic acid, (iii) the sulfhydryl group (-SH) in the side chain of cysteine amino acid residues, and (iv) the carbonyl group of carbohydrates in glycoproteins. The major methods include physical, irradiation-based, chemical and enzymatic crosslinking, among which the enzymatic method is the most expensive. Physical crosslinking methods involve temporary or reversible noncovalent or weak covalent bonds, whereas chemical crosslinking agents that crosslink proteins with relatively strong covalent bonds. Physical crosslinking of proteins is less costly and relatively less cytotoxic than chemical methods, but its crosslinking effect is relatively weak and has the potential to alter the properties of proteins. Another type of crosslinking reagents involves irradiation-based methods, including ultraviolet (UV) treatment, gamma irradiation and photochemical crosslinking techniques. Crosslinking reagents, also known as crosslinkers, contain at least two reactive groups to specific functional groups on proteins or other molecules. They have been used to assist in the determination of near-neighbor relationships, structures of proteins, solid-phase immobilization, hapten-carrier protein conjugation and molecular associations in cell membranes. Crosslinking reagents are also used in the preparation of antibody-enzyme conjugates, immunotoxins, and other labeled protein reagents.
Crosslinking reagents are selected on the basis of their properties, including chemical specificity, spacer arm length, water solubility and cell membrane permeability, homo-bifunctional or heterobifunctional reactive groups, cleavability, moieties that can be radiolabeled or labeled, and compatibility with the application. The length of the spacer arm determines the distance between potential reaction sites for crosslinking. Short spacer arms are often used in the intramolecular crosslinks, whereas crosslinkers containing long spacer arms are suitable for intermolecular crosslinks. Crosslinkers that are cleavable, non-cleavable, and have different spacer arm lengths are commonly used for complete analysis of protein structure. For different biochemistry needs, Amerigo Scientific offers a wide range of crosslinking reagents with different reactive groups and spacers of different lengths. The reactive groups on our crosslinkers include NHS and TFP ester, maleimide, dipyridyl disulfide (SPDP), bromoacetamide, etc. Linear and branched architectures are available. Our products are suitable for stabilizing structures in protein-protein, protein-peptide and protein-small molecule interactions, in immobilizing proteins onto a solid support, as well as for various peptide-nucleic acid and nucleic acid-nucleic acid conjugations.
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