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

研究成果: Chapter

摘要

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.

原文English
主出版物標題Methods in Molecular Biology
發行者Humana Press Inc.
頁面29-36
頁數8
DOIs
出版狀態Published - 2018

出版系列

名字Methods in Molecular Biology
1684
ISSN(列印)1064-3745

All Science Journal Classification (ASJC) codes

  • 分子生物學
  • 遺傳學

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