The label transfer approach incorporates crosslinking methodology to study protein-protein interactions by labeling proteins that interact with a protein of interest. This method can be used to discover novel interactions, confirm putative interactions suggested by other techniques, and investigate the protein complexes. In addition, the label transfer method can detect weak, or transient protein interactions that often fail to detect by co-immunoprecipitation, or Pull-Down method.
In a typical label transfer workflow, at first a bait protein reacts with a reactive moiety of a label transfer reagent (LTR) with a radioactive, fluorescent or biotin label; then labeled bait protein is then allowed to interact with prey protein in vitro to form a stable or transient complexes, then exposed to ultraviolet light to activate a photo-reactive group moiety on the crosslinker, which then covalently binds to prey proteins; the label transfer is completed by cleaving the crosslinker spacer arm to release the attached bait protein. The protein-protein interaction can be analyzed with multiple methods, including Westernblot, protein sequencing and mass spec analysis.
In previous research, fluorescent or radiolabelled aryl azides, carbenes or benzophenones are linked to hormones, peptides, or nucleic acids and used to label and explore previously unknown prey proteins. Using the label transfer approach has been widely applied to examine nucleic acid-protein interactions in this regard. Although most of the LTRs are valuable for direct labeling of specific protein domains, they are not generally useful for mapping of protein interaction networks, because the homing molecule attached to the cross-linker usually only directs cross-linking to the binding domain.
With updated versions of the label transfer strategy, Creative Proteomics can help to select suitable cross-linkers for you experimental goals, and carry out the consequent analysis for your exploration of protein-protein interactions of interest.
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