Two-Step Co-Immunoprecipitation (TIP) Enables Efficient and Highly Selective Isolation of Native Protein-Complexes.

Ruggero De Maria Marchiano, Tobias Longin Haas, Maria Rita Sciuto, Cecilia Valvo, Uwe Warnken, Martina Schnölzer, Lidia Brunetto, Alessandra Boe, Mauro Biffoni, Peter H. Krammer

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4 Citazioni (Scopus)

Abstract

Co-immunoprecipitation (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 co-precipitated 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 co-immunoprecipitations 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.
Lingua originaleEnglish
pagine (da-a)N/A-N/A
Numero di pagine38
RivistaMOLECULAR & CELLULAR PROTEOMICS
DOI
Stato di pubblicazionePubblicato - 2017

Keywords

  • proteomics

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