Investigation of neurotransmitter receptors in brain slices using cell surface biotinylation

Research output: Chapter in Book/Report/Conference proceedingChapter in a book

Abstract

Cell surface trafficking and endocytosis of neurotransmitter receptors are important regulatory mechanisms of neurotransmission. Biotinylation of plasma membrane proteins in brain slices allow their separation from those present in intracellular organelles. Membrane-impermeable, thiol-cleavable amine-reactive biotinylation reagents (e.g. EZ-link sulfo-NH-SS-biotin) form a stable covalent linkage with primary amino groups of surface exposed proteins. Following homogenisation of brain slices and solubilisation of membranes, biotin-labelled proteins can be isolated with avidin or streptavidin linked to agarose beads. Bound biotinylated proteins are released from avidin or streptavidin in the presence of reducing agents (e.g. glutathione or β-mercaptoethanol). Quantitative differences in the molecular composition of biotin-labelled (surface) and unlabelled (intracellular) protein fractions can be analysed using immunoblotting with target protein specific antibodies. While many variations of this procedure exist in the literature, in this chapter we describe the biotinylation protocol that we have applied for the investigation of quantitative changes in the cell surface expression and internalisation of ionotropic glutamate receptors in acute brain slices.
Original languageEnglish
Title of host publicationReceptor and Ion Channel Detection in the Brain
Subtitle of host publicationMethods and Protocols
EditorsRafael Lujan, Francisco Ciruela
PublisherSpringer
Pages39-48
Number of pages10
ISBN (Electronic)9781493930647
ISBN (Print)9781493930630
DOIs
Publication statusPublished - 2 Feb 2016

Publication series

NameNeuromethods
PublisherSpringer New York
Volume110
ISSN (Print)0893-2336

Keywords

  • antibodies
  • avidin
  • biotinylation
  • cell surface expression
  • endocytosis
  • internalisation
  • targeting
  • trafficking

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