Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)
| Autor: | Sean P. Kennedy, Jeremy Z. R. Han, Mary Iconomou, Darren N. Saunders, Robert F. Shearer, Jordan F. Hastings, David R. Croucher |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Proteomics General Immunology and Microbiology Chemistry General Chemical Engineering General Neuroscience Proteins Computational biology Protein complex assembly General Biochemistry Genetics and Molecular Biology Fluorescence Protein–protein interaction Complementation 03 medical and health sciences Bimolecular fluorescence complementation 030104 developmental biology Affinity chromatography Protein-fragment complementation assay Multiprotein Complexes Humans Protein Interaction Domains and Motifs Signal transduction Immunology and Infection Signal Transduction |
| Zdroj: | J Vis Exp |
| Popis: | The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes. Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques. |
| Databáze: | OpenAIRE |
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