Two-Step Co-Immunoprecipitation (TIP)

Maria Rita Sciuto, Valeria Coppola, Gioacchin Iannolo, Ruggero De Maria, Tobias L. Haas

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article numbere80
JournalCurrent Protocols in Molecular Biology
Volume125
Issue number1
DOIs
Publication statusPublished - Jan 1 2019

Fingerprint

Immunoprecipitation
Proteins
Burkitt Lymphoma
Nucleic Acids
Chromatin
Transcription Factors
T-Lymphocytes

Keywords

  • antibody
  • co-immunoprecipitation
  • protein-nucleic acid interaction
  • protein-protein interaction

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Two-Step Co-Immunoprecipitation (TIP). / Sciuto, Maria Rita; Coppola, Valeria; Iannolo, Gioacchin; De Maria, Ruggero; Haas, Tobias L.

In: Current Protocols in Molecular Biology, Vol. 125, No. 1, e80, 01.01.2019.

Research output: Contribution to journalArticle

@article{94113f2e079243008ae49ea860038ed1,
title = "Two-Step Co-Immunoprecipitation (TIP)",
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.",
keywords = "antibody, co-immunoprecipitation, protein-nucleic acid interaction, protein-protein interaction",
author = "Sciuto, {Maria Rita} and Valeria Coppola and Gioacchin Iannolo and {De Maria}, Ruggero and Haas, {Tobias L.}",
year = "2019",
month = "1",
day = "1",
doi = "10.1002/cpmb.80",
language = "English",
volume = "125",
journal = "Current Protocols in Molecular Biology",
issn = "1934-3639",
publisher = "John Wiley and Sons Inc.",
number = "1",

}

TY - JOUR

T1 - Two-Step Co-Immunoprecipitation (TIP)

AU - Sciuto, Maria Rita

AU - Coppola, Valeria

AU - Iannolo, Gioacchin

AU - De Maria, Ruggero

AU - Haas, Tobias L.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - antibody

KW - co-immunoprecipitation

KW - protein-nucleic acid interaction

KW - protein-protein interaction

UR - http://www.scopus.com/inward/record.url?scp=85055957660&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85055957660&partnerID=8YFLogxK

U2 - 10.1002/cpmb.80

DO - 10.1002/cpmb.80

M3 - Article

VL - 125

JO - Current Protocols in Molecular Biology

JF - Current Protocols in Molecular Biology

SN - 1934-3639

IS - 1

M1 - e80

ER -