Two‐Step Co‐Immunoprecipitation (TIP). Issue 1 (30th October 2018)

• Record Type: Journal Article
• Authors: Sciuto, Maria Rita; Coppola, Valeria; Iannolo, Gioacchin; De Maria, Ruggero; Haas, Tobias L.
• Editors: Ausubel, Frederick M.; Brent, Roger; Kingston, Robert E.; Moore, David D.; Seidman, J.G.; Smith, John A.; Struhl, Kevin
• Is Part Of: Current protocols in molecular biology. Volume 125:Issue 1(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-10-30
• Subjects: antibody -- co‐immunoprecipitation -- protein‐nucleic acid interaction -- protein‐protein interaction; Molecular biology -- Technique -- Periodicals; Molecular biology -- Laboratory manuals; Molecular Biology -- methods; Biologie moléculaire -- Technique; Biologie moléculaire -- Manuels de laboratoire; Molecular biology; Molecular biology -- Technique; Laboratory Manual; Electronic reference sources; Laboratory manuals; 572.8028
• DOI: 10.1002/cpmb.80
• Languages: English
• ISSNs: 1934-3639

Abstract

Abstract: In the past few decades, numerous approaches have been developed to investigate protein‐protein and protein‐nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co‐immunoprecipitation (co‐IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two‐step co‐immunoprecipitation (TIP) technique. As compared to standard co‐IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein‐protein complex from Burkitt lymphoma cells and from primary human CD4 + T cells. In addition, this unit describes an application of TIP for the isolation of transcription‐factor‐bound chromatin. © 2018 by John Wiley & Sons, Inc.