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

Producción científica: Chapter › revisión exhaustiva

5 Citas (Scopus)

Resumen

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.

Idioma original	English
Título de la publicación alojada	Methods in Molecular Biology
Páginas	337-348
Número de páginas	12
DOI	https://doi.org/10.1007/978-1-0716-0814-2_20
Estado	Published - 2021

Serie de la publicación

Nombre	Methods in Molecular Biology
Volumen	2187
ISSN (versión impresa)	1064-3745
ISSN (versión digital)	1940-6029

Nota bibliográfica

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

Financiación

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.

Financiadores	Número del financiador
National Science Foundation Arctic Social Science Program	1615874
National Science Foundation Arctic Social Science Program
National Institutes of Health (NIH)
Institute of Neurological Disorders and Stroke National Advisory Neurological Disorders and Stroke Council	R01NS095215
Institute of Neurological Disorders and Stroke National Advisory Neurological Disorders and Stroke Council
University of Kentucky

ASJC Scopus subject areas

Molecular Biology
Genetics

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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.",

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AU - Qin, Haiyan

AU - Elsherbini, Ahmed

AU - Roush, Emily A.

AU - Crivelli, Simone M.

AU - Spassieva, Stefka D.

AU - Bieberich, Erhard

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AB - 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.

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KW - Cross-linking

KW - Immunocytochemistry

KW - Proximity ligation assay (PLA)

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

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Financiación

ASJC Scopus subject areas

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