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| Item Type: | Article |
|---|---|
| Title: | DULIP: A dual luminescence-based co-immunoprecipitation assay for interactome mapping in mammalian cells |
| Creators Name: | Trepte, P. and Buntru, A. and Klockmeier, K. and Willmore, L. and Arumughan, A. and Secker, C. and Zenkner, M. and Brusendorf, L. and Rau, K. and Redel, A. and Wanker, E.E. |
| Abstract: | Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening as well as for the validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns. |
| Keywords: | Systematic Protein-Protein Interaction Screening, Luminescence Normalization, Quantitative Interaction Score and Quantification of Interaction Strength, Detection of Low- and High-Affinity Interactions, Disease-Mutation Detection, Animals |
| Source: | Journal of Molecular Biology |
| ISSN: | 0022-2836 |
| Publisher: | Elsevier |
| Volume: | 427 |
| Number: | 21 |
| Page Range: | 3375-3388 |
| Date: | 23 October 2015 |
| Official Publication: | https://doi.org/10.1016/j.jmb.2015.08.003 |
| PubMed: | View item in PubMed |
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