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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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
Pages (from-to)	337-348
Number of pages	12
Journal	Methods in Molecular Biology
Volume	2187
DOIs	https://doi.org/10.1007/978-1-0716-0814-2_20
Publication status	Published - 2021

Keywords

Cells, Cultured
Ceramides/analysis
Cross-Linking Reagents/chemistry
HEK293 Cells
Humans
Ligases/metabolism
Membrane Lipids/analysis
Membrane Microdomains/chemistry
Membrane Proteins/analysis
Staining and Labeling/methods

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

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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 = "Cells, Cultured, Ceramides/analysis, Cross-Linking Reagents/chemistry, HEK293 Cells, Humans, Ligases/metabolism, Membrane Lipids/analysis, Membrane Microdomains/chemistry, Membrane Proteins/analysis, Staining and Labeling/methods",

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.",

year = "2021",

doi = "10.1007/978-1-0716-0814-2_20",

language = "English",

volume = "2187",

pages = "337--348",

journal = "Methods in Molecular Biology",

issn = "1064-3745",

publisher = "Springer Nature Switzerland AG",

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TY - JOUR

T1 - Cross-Link/Proximity Ligation Assay for Visualization of Lipid and Protein Complexes in Lipid Rafts

AU - Tripathi, Priyanka

AU - Zhu, Zhihui

AU - Qin, Haiyan

AU - Elsherbini, Ahmed

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

Y1 - 2021

N2 - 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.

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.

KW - Cells, Cultured

KW - Ceramides/analysis

KW - Cross-Linking Reagents/chemistry

KW - HEK293 Cells

KW - Humans

KW - Ligases/metabolism

KW - Membrane Lipids/analysis

KW - Membrane Microdomains/chemistry

KW - Membrane Proteins/analysis

KW - Staining and Labeling/methods

U2 - 10.1007/978-1-0716-0814-2_20

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

M3 - Article

C2 - 32770517

SN - 1064-3745

VL - 2187

SP - 337

EP - 348

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -

Cross-Link/Proximity Ligation Assay for Visualization of Lipid and Protein Complexes in Lipid Rafts

Abstract

Keywords

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