UNIT 5.30 A Protein Cross-Linking Assay for Measuring Cell Surface Expression of Glutamate Receptor Subunits in the Rodent Brain After In Vivo Treatments
Published Online: 1 APR 2012
Copyright © 2012 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Neuroscience
How to Cite
Boudreau, A. C., Milovanovic, M., Conrad, K. L., Nelson, C., Ferrario, C. R. and Wolf, M. E. 2012. A Protein Cross-Linking Assay for Measuring Cell Surface Expression of Glutamate Receptor Subunits in the Rodent Brain After In Vivo Treatments. Current Protocols in Neuroscience. 59:5.30:5.30.1–5.30.19.
- Published Online: 1 APR 2012
- Published Print: APR 2012
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. Curr. Protoc. Neurosci. 59:5.30.1-5.30.19. © 2012 by John Wiley & Sons, Inc.
- cell surface expression;
- glutamate receptors;
- protein cross-linking;
- receptor trafficking