Monitoring changes in the abundance of endogenously expressed ATP-sensitive potassium (KATP) channels in the plasma membrane using surface biotinylation

Jing Syuna Ruan, Pei Chun Chen

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages29-36
Number of pages8
DOIs
Publication statusPublished - 2018 Jan 1

Publication series

NameMethods in Molecular Biology
Volume1684
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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