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 complexes resulting from these interactions. In this article, we describe a two-step coimmunoprecipitation (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.

Sciuto, M. R., Coppola, V., Iannolo, G., De Maria Marchiano, R., Haas, T. L., Two-Step Co-Immunoprecipitation (TIP), <<CURRENT PROTOCOLS IN MOLECULAR BIOLOGY>>, 2018; 125 (1): e80-e100. [doi:10.1002/cpmb.80] [http://hdl.handle.net/10807/127895]

Two-Step Co-Immunoprecipitation (TIP)

De Maria Marchiano, Ruggero;Haas, Tobias Longin
2019

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 complexes resulting from these interactions. In this article, we describe a two-step coimmunoprecipitation (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.
2019
Inglese
Sciuto, M. R., Coppola, V., Iannolo, G., De Maria Marchiano, R., Haas, T. L., Two-Step Co-Immunoprecipitation (TIP), <<CURRENT PROTOCOLS IN MOLECULAR BIOLOGY>>, 2018; 125 (1): e80-e100. [doi:10.1002/cpmb.80] [http://hdl.handle.net/10807/127895]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10807/127895
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