Multiplex IP-FCM (immunoprecipitation-flow cytometry): Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes

Anya T. Bida, Diana Gil, Adam G. Schrum

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

There is significant interest in the development of methods with the potential to increase access to 'the interactome' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.

Original languageEnglish (US)
Pages (from-to)154-160
Number of pages7
JournalMethods
Volume56
Issue number2
DOIs
StatePublished - Feb 2012

Fingerprint

Multiprotein Complexes
Flow cytometry
Immunoprecipitation
Flow Cytometry
Guidelines
Proteins
Microspheres
Assays
Data acquisition
Antibodies

Keywords

  • Flow cytometry
  • IP-FCM
  • Multiplex
  • Multiprotein complex
  • Protein-protein interaction

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Multiplex IP-FCM (immunoprecipitation-flow cytometry) : Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes. / Bida, Anya T.; Gil, Diana; Schrum, Adam G.

In: Methods, Vol. 56, No. 2, 02.2012, p. 154-160.

Research output: Contribution to journalArticle

@article{c55c1b37a47d40a3aff544c4b9f21cfe,
title = "Multiplex IP-FCM (immunoprecipitation-flow cytometry): Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes",
abstract = "There is significant interest in the development of methods with the potential to increase access to 'the interactome' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.",
keywords = "Flow cytometry, IP-FCM, Multiplex, Multiprotein complex, Protein-protein interaction",
author = "Bida, {Anya T.} and Diana Gil and Schrum, {Adam G.}",
year = "2012",
month = "2",
doi = "10.1016/j.ymeth.2011.09.005",
language = "English (US)",
volume = "56",
pages = "154--160",
journal = "ImmunoMethods",
issn = "1046-2023",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Multiplex IP-FCM (immunoprecipitation-flow cytometry)

T2 - Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes

AU - Bida, Anya T.

AU - Gil, Diana

AU - Schrum, Adam G.

PY - 2012/2

Y1 - 2012/2

N2 - There is significant interest in the development of methods with the potential to increase access to 'the interactome' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.

AB - There is significant interest in the development of methods with the potential to increase access to 'the interactome' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.

KW - Flow cytometry

KW - IP-FCM

KW - Multiplex

KW - Multiprotein complex

KW - Protein-protein interaction

UR - http://www.scopus.com/inward/record.url?scp=84858451531&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84858451531&partnerID=8YFLogxK

U2 - 10.1016/j.ymeth.2011.09.005

DO - 10.1016/j.ymeth.2011.09.005

M3 - Article

C2 - 21945581

AN - SCOPUS:84858451531

VL - 56

SP - 154

EP - 160

JO - ImmunoMethods

JF - ImmunoMethods

SN - 1046-2023

IS - 2

ER -