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Potassium Channels pp 29–36Cite as

Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1684))

Abstract

The conductance of KATP channel activity can be regulated by gating and/or surface expression. Gating analysis is usually performed by electrophysiological recording. Analysis of surface KATP channel expression levels requires cell fractionation, protein separation, and quantification. Cell fractionation involves time-consuming high-speed centrifugation steps and skilled techniques for taking out specific layers. Moreover, contamination of intracellular membranes can confound results. Although commercial kits have been developed to make it easier for scientists, qualities of these kits vary which can give rise to variable results. Detection of membrane proteins using surface biotinylation technique consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pulldown. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes, and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control). This alternative method for detecting expression of surface protein is relatively easy in steps and more economical in comparison to other methods such as cell fractionation.

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Acknowledgment

This work received financial support from the Ministry of Science and Technology (MOST103-2320-B-006-005-MY2 and MOST105-2628-B-006-006-MY3) to P.C.C.

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Authors and Affiliations

  1. Department of Physiology, College of Medicine, National Cheng Kung University, No. 1, University Road, Tainan, 701, Taiwan

    Jing-Syuna Ruan & Pei-Chun Chen

Authors
  1. Jing-Syuna Ruan
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  2. Pei-Chun Chen
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Corresponding author

Correspondence to Pei-Chun Chen .

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Editors and Affiliations

  1. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA

    Show-Ling Shyng

  2. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA

    Francis I. Valiyaveetil

  3. The Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA

    Matt Whorton

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Ruan, JS., Chen, PC. (2018). Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation. In: Shyng, SL., Valiyaveetil, F., Whorton, M. (eds) Potassium Channels. Methods in Molecular Biology, vol 1684. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7362-0_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7362-0_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7361-3

  • Online ISBN: 978-1-4939-7362-0

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