Two-Step Coimmunoprecipitation (TIP) enables efficient and highly selective isolation of native protein complexes

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

Research output: Contribution to journalArticle

Abstract

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 bench-marked TIP for the identification of CD95/FAS-interact-ing 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.

Original languageEnglish
Pages (from-to)993-1009
Number of pages17
JournalMolecular and Cellular Proteomics
Volume17
Issue number5
DOIs
Publication statusPublished - May 1 2018

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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