An Overview of Application of Protein Cross-linking Agent

  • Protein cross-linking agents are a class of small molecule compounds that have 2 or more reactive ends for special groups (-NH2, -COOH, -HS, etc.) and can be coupled to 2 or more molecules, respectively, so that these molecules bind together. In the 1970s, people generally used glutaraldehyde as a protein cross-linking agent to connect antibodies and indicators (such as enzymes), but its disadvantage is that the cross-linking group is random, and it is easy to form messy multimers. In the 1980s, more selective specific crosslinking agents, such as NHS (for -COOH) and maleimide (for -HS), were more widely used in life science research. The clever use of cross-linking agents can achieve unexpected gains in protein interaction research, immunology, cancer treatment and other fields.



    Protein crosslinking agents have been widely used in various branches of life science research internationally.

    Research on protein-protein interaction

    Most protein-protein binding interactions are transient, and the components in the interaction complex can be stabilized or permanently linked by cross-linking methods, thereby helping to identify these transient contacts. Once the interacting components are covalently coupled, other steps can be used to prepare the analysis sample while maintaining the original interacting complex.


    Antibodies that recognize tumor surface antigens are covalently linked to drugs to achieve targeted therapy, thereby reducing the killing of healthy cells by traditional chemotherapy methods.


    Carrier protein and hapten connection

    Haptens are only reactive and not immunogenic. And a polypeptide sequence of the virus coating protein can be connected with the carrier protein to make a vaccine to stimulate the immune response. In addition, a monoclonal antibody can also be prepared by this method, and the monoclonal antibody can be used to return to find the antigen binding site on the macromolecular protein. In the operation of connecting the two, the lower hapten-carrier protein molar ratio will bring higher antibody affinity.


    In addition, linking the hapten to the amino acid in the middle of the carrier protein has a higher titer than the antibody produced when it is linked to the terminal.


    Solidification of proteins or other molecules

    Covalently bind proteins to solid substrates such as magnetic beads, 96-well plates, glass, etc. to reduce losses during elution

    Antibody labeling, label transfer

    The antibody is linked to an enzyme, fluorophore or biotin for quantitative measurement or localization observation. Before antibody labeling and modification, it is necessary to purify with immobilized antigen. In order to avoid the site where the antibody binds to the antigen being blocked, it is important to select the amino acid at the appropriate position on the antibody heavy chain. The labeling enzyme can be attached to a disulfide bond formed by opening the hinge region of the antibody, or to a non-specific fragment of the antibody.


    In addition, the antibody can also be digested to obtain an antigen-binding fragment, and then the labeling group is connected to the antigen-binding fragment. The advantage of this method is that it reduces the interference of Fc fragments in the ELISA reaction, and also increases the membrane permeability of this complex, making it more suitable for labeling living cells. The picture on the left is the binding of the labeling group to the thiol group generated by the opening of the disulfide bond in the hinge region.