Abstract
This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody capture. Firstly, a primary antibody is captured by an extracellular epitope presented by the target membrane protein and is subsequently internalized. Secondly, the primary antibody-labelled protein is recycled back to the membrane where it is captured by a probe-conjugated secondary antibody. In the qualitative assay, the probe is a fluorophore, which can be imaged by fluorescence microscopy. In the quantitative assay, the probe is horse-radish peroxidase (HRP) and enzyme activity can be assayed by chemiluminescence.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
van der Sluijs P, Hull M, Webster P, Mâle P, Goud B, Mellman I (1992) The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell 70:729–740
Wilcke M, Johannes L, Galli T, Mayau V, Goud B, Salamero J (2000) Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network. J Cell Biol 151:1207–1220
Gokool S, Tattersall D, Seaman MN (2007) EHD1 interacts with retromer to stabilize SNX1 tubules and facilitate endosome-to-Golgi retrieval. Traffic 8:1873–1886
Maxfield FR, McGraw TE (2004) Endocytic recycling. Nat Rev Mol Cell Biol 5:121–132
Grant BD, Donaldson JG (2009) Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol 10:597–608
Park M, Penick EC, Edwards JG, Kauer JA, Ehlers MD (2004) Recycling endosomes supply AMPA receptors for LTP. Science 305:1972–1975
Yu SS, Lefkowitz RJ, Hausdorff WP (1993) Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization. J Biol Chem 268:337–341
Shewan AM, van Dam EM, Martin S, Luen TB, Hong W, Bryant NJ, James DE (2003) GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif. Mol Biol Cell 14:973–986
Manna PT, Smith AJ, Taneja TK, Howell GJ, Lippiat JD, Sivaprasadarao A (2009) Constitutive endocytic recycling and protein kinase C-mediated lysosomal degradation control K(ATP) channel surface density. J Biol Chem 285:5963–5973
Smith AJ, Sivaprasadarao A (2008) Investigation of K(ATP) channel endocytosis by immunofluorescence. In: Lippiat JD (ed) Potassium channels, methods in molecular biology, vol 491. Humana Press, New York, pp 69–77
Smith AJ, Sivaprasadarao A (2008) Chemiluminescence assays to investigate membrane expression and clathrin-mediated endocytosis of K(ATP) channels. In: Lippiat JD (ed) Potassium channels, methods in molecular biology, vol 491. Humana Press, New York, pp 63–68
Zerangue N, Schwappach B, Jan YN, Jan LY (1999) A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 22:537–548
Acknowledgement
The author wishes to thank Prof. A. Sivaprasadarao for advice on manuscript preparation and the MRC for funding support.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Cockcroft, C.J. (2013). Imaging and Quantification of Recycled KATP Channels. In: Gamper, N. (eds) Ion Channels. Methods in Molecular Biology, vol 998. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-351-0_18
Download citation
DOI: https://doi.org/10.1007/978-1-62703-351-0_18
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-350-3
Online ISBN: 978-1-62703-351-0
eBook Packages: Springer Protocols