When two or more proteins have specific affinity for one another to assemble complexes in biological systems, most in vivo protein-protein interactions are transient, for cellular signaling or metabolic functions. Capturing or freezing these brief contacts to study which proteins are involved and how they interact is a significant part of proteomic research. Crosslinking reagents, or crosslinkers, provide the analytical solution to capturing protein-protein complexes by covalently binding them together as they interact, to freeze even transient , weak interactions for consequent isolation and characterization. The crosslinking can be performed in vivo or in vitro. The benefit of in vivo crosslinking is that the protein-protein interaction can be captured in its native environment, to avoid false positive interactions and loss of complex stability during sample preparation. Hydrophobic, lipid-soluble crosslinkers are expected to capture target proteins within or across cell membranes, while hydrophilic, water-soluble crosslinkers are used to crosslink cell surface proteins.

Although in vivo crosslinking can yield physiologically relevant and stable complexes for consequent analysis, although the reaction conditions cannot be strictly controlled and crosslinkers may react with various proteins containing functional groups against which crosslinkers can specifically react.

Contrast to in vivo crosslinking, in vitro crosslinking can offer better targeted crosslinking, because reaction conditions can be tightly regulated, such as the pH, temperature, and concentration of reactants, which results in higher resolution of protein-protein interactions. Meanwhile, in vitro crosslinking allow to modify interacting proteins, and hydrophobic and hydrophilic crosslinking reagents are commercial available. The problems involved in vitro crosslinking is that protein complexes are not assembled in non-physiological conditions; and dissolving plasma membranes can disrupt, and even break protein-protein and protein-membrane interactions.

Because lots of crosslinking reagents are commercially available for many different applications, the key determinant in deciding to use in vivo or in vitro crosslinking is the target protein, specifically depending on cellular location & interaction stability of protein complexes. Our scientists in Creative Proteomics are glad to discuss the experimental details with you, and provide rapid and reliable analytical services of protein-protein interactions.