Cross-link/proximity ligation assay for visualization of lipid and protein complexes in lipid rafts

Priyanka Tripathi; Zhihui Zhu; Haiyan Qin; Ahmed Elsherbini; Emily A. Roush; Simone M. Crivelli; Stefka D. Spassieva; Erhard Bieberich

doi:10.1007/978-1-0716-0814-2_20

Cross-link/proximity ligation assay for visualization of lipid and protein complexes in lipid rafts

Priyanka Tripathi, Zhihui Zhu, Haiyan Qin, Ahmed Elsherbini, Emily A. Roush, Simone M. Crivelli, Stefka D. Spassieva, Erhard Bieberich

Physiology

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

2 Scopus citations

Abstract

The detection of protein complexes by coimmunoprecipitation or two-hybrid analysis is often limited to cytosolic and soluble proteins, while interaction between membrane proteins or proteins and lipids is hampered by solubilization artefacts or absence of appropriate antibodies to detect a complex. More recently, the proximity ligation assay (PLA) using antibodies for in situ detection of protein complexes in cells and cross-linkable lipid analogs that can be endowed with molecular tags for pull-down assyas were techniques utilized to identify and monitor interaction between proteins and lipids. We have developed a novel technique termed “cross-link/PLA” combining a cross-linkable ceramide analog with PLA and anti-ceramide antibody to visualize lipid–protein complexes in ceramide-rich platforms (CRPs), a particular type of lipid raft. This chapter will discuss experimental protocols and data analysis to use cross-link/PLA for detection and visualization of lipid–protein complexes in CRPs and other types of lipid rafts.

Original language	English
Title of host publication	Methods in Molecular Biology
Pages	337-348
Number of pages	12
DOIs	https://doi.org/10.1007/978-1-0716-0814-2_20
State	Published - 2021

Publication series

Name	Methods in Molecular Biology
Volume	2187
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Bibliographical note

Funding Information:
This work was in part supported by NIH grant R01NS095215 and NSF grant 1615874. The authors thank Chair Dr. Alan Daugherty, the Department of Physiology, College of Medicine, University of Kentucky, for institutional support.

Publisher Copyright:
© 2021, Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

Biorthogonal analog
Ceramide-rich platform (CRP)
Click chemistry
Cross-linking
Immunocytochemistry
Proximity ligation assay (PLA)

ASJC Scopus subject areas

Molecular Biology
Genetics

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10.1007/978-1-0716-0814-2_20

Cite this

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title = "Cross-link/proximity ligation assay for visualization of lipid and protein complexes in lipid rafts",

abstract = "The detection of protein complexes by coimmunoprecipitation or two-hybrid analysis is often limited to cytosolic and soluble proteins, while interaction between membrane proteins or proteins and lipids is hampered by solubilization artefacts or absence of appropriate antibodies to detect a complex. More recently, the proximity ligation assay (PLA) using antibodies for in situ detection of protein complexes in cells and cross-linkable lipid analogs that can be endowed with molecular tags for pull-down assyas were techniques utilized to identify and monitor interaction between proteins and lipids. We have developed a novel technique termed “cross-link/PLA” combining a cross-linkable ceramide analog with PLA and anti-ceramide antibody to visualize lipid–protein complexes in ceramide-rich platforms (CRPs), a particular type of lipid raft. This chapter will discuss experimental protocols and data analysis to use cross-link/PLA for detection and visualization of lipid–protein complexes in CRPs and other types of lipid rafts.",

keywords = "Biorthogonal analog, Ceramide-rich platform (CRP), Click chemistry, Cross-linking, Immunocytochemistry, Proximity ligation assay (PLA)",

author = "Priyanka Tripathi and Zhihui Zhu and Haiyan Qin and Ahmed Elsherbini and Roush, {Emily A.} and Crivelli, {Simone M.} and Spassieva, {Stefka D.} and Erhard Bieberich",

note = "Funding Information: This work was in part supported by NIH grant R01NS095215 and NSF grant 1615874. The authors thank Chair Dr. Alan Daugherty, the Department of Physiology, College of Medicine, University of Kentucky, for institutional support. Publisher Copyright: {\textcopyright} 2021, Springer Science+Business Media, LLC, part of Springer Nature.",

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AU - Roush, Emily A.

AU - Crivelli, Simone M.

AU - Spassieva, Stefka D.

AU - Bieberich, Erhard

N1 - Funding Information: This work was in part supported by NIH grant R01NS095215 and NSF grant 1615874. The authors thank Chair Dr. Alan Daugherty, the Department of Physiology, College of Medicine, University of Kentucky, for institutional support. Publisher Copyright: © 2021, Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2021

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KW - Cross-linking

KW - Immunocytochemistry

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Cross-link/proximity ligation assay for visualization of lipid and protein complexes in lipid rafts

Abstract

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Keywords

ASJC Scopus subject areas

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