Two-Step Coimmunoprecipitation (TIP) Enables Efficient and Highly Selective Isolation of Native Protein Complexes
| Autor: | Tobias L. Haas, Mauro Biffoni, Maria Rita Sciuto, Alessandra Boe, Lidia Brunetto, Uwe Warnken, Martina Schnölzer, Cecilia Valvo, Peter H. Krammer, Ruggero De Maria |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
CD4-Positive T-Lymphocytes Proteomics Chromatin Immunoprecipitation Immunoprecipitation Receptors Cytoplasmic and Nuclear Apoptosis Plasma protein binding Karyopherins Biochemistry Analytical Chemistry Cell Line 03 medical and health sciences Gene Knockdown Techniques Humans Biotinylation Amino Acid Sequence fas Receptor Molecular Biology Peptide sequence Settore MED/06 - ONCOLOGIA MEDICA biology Chemistry Technological Innovation and Resources Antibodies Monoclonal Reproducibility of Results Protein Phosphatase 2C 030104 developmental biology Polyclonal antibodies Multiprotein Complexes biology.protein Biophysics Chromatin immunoprecipitation Protein Binding |
| Zdroj: | Molecularcellular proteomics : MCP. 17(5) |
| ISSN: | 1535-9484 |
| Popis: | Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4(+) T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions. |
| Databáze: | OpenAIRE |
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