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Dissecting multi-protein signaling complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Abstract

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.

Type Journal
ISBN 1940-087x
Authors Hastings, J. F.; Han, J. Z. R.; Shearer, R. F.; Kennedy, S. P.; Iconomou, M.; Saunders, D. N.; Croucher, D. R.
Responsible Garvan Author A/Prof David Croucher
Publisher Name Jove-Journal of Visualized Experiments
Published Date 2018-06-15
Published Issue 136
Published Pages e57109
Status Always Electronic
DOI 10.3791/57109
URL link to publisher's version https://www.ncbi.nlm.nih.gov/pubmed/29985350
OpenAccess link to author's accepted manuscript version https://publications.gimr.garvan.org.au/open-access/14763