Mapping protein-protein interactions of protein complexes is a central problem in biology. Traditional methods for detecting protein interactions such as yeast two-hybrid, pull down and immunoprecipitation have their own limitations. Yeast two-hybrid can reveal direct interactions between proteins, and even discover unknown interactions through large-scale screening, but yeast cells may not provide suitable interaction conditions for heterologously expressed proteins of other species. Affinity chromatography and co-immunoprecipitation techniques have relatively low throughput, and it is sometimes difficult to distinguish background-binding proteins from specific binding proteins, and direct and indirect interactions are often difficult to distinguish. In addition, these two methods are difficult to obtain effective information for transient, weak interactions, such as loosely changing protein complexes during signal transduction.
Cross-linking and label transfer will no doubt be important in this regard and hopefully the evolution of these technologies will lead to better reagents and techniques available to biochemists for unraveling enzymatic structure and function. Label transfer can be used to discover new interactions, as well as to validate protein interactions detected by other techniques, and can be applied to the study of protein complexes. In addition, label transfer methods can detect weak and transient protein interactions that are not detected by traditional methods.
Photoaffinity labeling techniques that can be used to examine protein structure and function can yield hundreds of examples. Usually, fluorescent or radioactively labeled arylazides are tethered to carbene, benzophenones, hormones, peptides or nucleic acids, etc., can be used to label and explore unknown target proteins. Label transfer methods have been widely used for nucleic acid-protein interaction detection.
In practice, label transfer reagents (LTR) with a radioactive, fluorescent or biotin label are first tethered to a target protein of interest. After this modified protein is reconstituted into a stable or transient complex in vitro, the radio-iodinated photo-probe is activated with ultraviolet light resulting in a cross-link (formed by a radical mechanism) to the nearest protein neighbor. Finally, the linker arm connecting the two proteins is severed, thus revealing the protein−protein interaction by label transfer.
These label transfer reagents (LTRs) contain three modular components : (1) an ATP-competitive kinase inhibitor that targets the labeling reagent to a kinase target of interest; (2) a photo-crosslinker, which allows covalent labeling of any proteins that are in proximity to the LTR; and (3) an orthogonal chemical alkyne tag for visualization and/or purification of proteins that have been labeled.
Label transfer reagents (LTRs) for the identification of the interacting partners of protein kinases.
Using the latest label transfer strategy, Creative Proteomics can select appropriate cross-linking reagents according to your experimental goals, and perform subsequent analysis to help you explore target protein-protein interactions.
Able to effectively monitor weak or transient interactions in vivo
Able to study the structure of soluble or inaccessible proteins
Provide valid kinetic data
Increase the detection sensitivity
Short experiment period
Creative Proteomics is an international biotechnology company dedicated to the study of intermolecular interactions and other related fields. In order to provide customers with higher quality and efficient services, we have established many powerful technical platforms to support our services, such as the upgraded pull-down assay platform. Customers can complete a large number of advanced experiments and projects on our platform.