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

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

4 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 languageEnglish
Title of host publicationMethods in Molecular Biology
Pages337-348
Number of pages12
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2187
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

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

Fingerprint

Dive into the research topics of 'Cross-link/proximity ligation assay for visualization of lipid and protein complexes in lipid rafts'. Together they form a unique fingerprint.

Cite this