Projects per year
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.
|Title of host publication||Receptor and Ion Channel Detection in the Brain|
|Subtitle of host publication||Methods and Protocols|
|Editors||Rafael Lujan, Francisco Ciruela|
|Number of pages||10|
|Publication status||Published - 2 Feb 2016|
|Publisher||Springer New York|
- cell surface expression
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- 2 Finished
Development of biotin-tagged affinity ligands and fluorophore-conjugated probes for the study of native kainate receptors
1/03/13 → 1/03/16
DISTRIBUTION AND MOLECULAR ORGANISATION OF KAINATE RECEPTORS IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM
1/12/07 → 1/12/11