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Imaging and quantification of recycled KATP channels
1Faculty of Biological Sciences, School of Biomedical Sciences, University of Leeds, Leeds, UK.
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.
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.
