Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 136, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/57109
Keywords
Immunology and Infection; Issue 136; Protein-protein interactions; Proteomics; Interactomics; BiCAP; Signal Transduction; Bimolecular fluorescence complementation; Nanobody
Categories
Funding
- Cancer Institute NSW [13/FRL/1-02, 09/CDF/2-39]
- NHMRC [GNT1052963]
- Science Foundation Ireland [11/SIRG/B2157]
- NSW Office of Science and Medical Research
- Guest Family Fellowship
- Mostyn Family Foundation
- Australian Postgraduate Award
- Science Foundation Ireland (SFI) [11/SIRG/B2157] Funding Source: Science Foundation Ireland (SFI)
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available