A protein cross-linking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments

Amy C. Boudreau, Mike Milovanovic, Kelly L. Conrad, Christopher Nelson, Carrie R. Ferrario, Marina Wolf

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface-expressed proteins are covalently cross-linked using the membrane-impermeable, bifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross-linking.We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants.

Original languageEnglish (US)
Article number5.30
JournalCurrent Protocols in Neuroscience
Volume1
Issue numberSUPPL.59
DOIs
StatePublished - Apr 1 2012
Externally publishedYes

Fingerprint

Glutamate Receptors
Rodentia
Brain
Biotinylation
Proteins
Neurotransmitter Receptor
AMPA Receptors
Immunoblotting
Synaptic Transmission
Polyacrylamide Gel Electrophoresis
Membrane Proteins
Molecular Weight
Costs and Cost Analysis
Membranes
Antibodies

Keywords

  • BS
  • Cell surface expression
  • Glutamate receptors
  • Protein cross-linking
  • Receptor trafficking

ASJC Scopus subject areas

  • Neuroscience(all)
  • Medicine(all)

Cite this

A protein cross-linking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments. / Boudreau, Amy C.; Milovanovic, Mike; Conrad, Kelly L.; Nelson, Christopher; Ferrario, Carrie R.; Wolf, Marina.

In: Current Protocols in Neuroscience, Vol. 1, No. SUPPL.59, 5.30, 01.04.2012.

Research output: Contribution to journalArticle

Boudreau, Amy C. ; Milovanovic, Mike ; Conrad, Kelly L. ; Nelson, Christopher ; Ferrario, Carrie R. ; Wolf, Marina. / A protein cross-linking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments. In: Current Protocols in Neuroscience. 2012 ; Vol. 1, No. SUPPL.59.
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