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 language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc. |
| Pages | 29-36 |
| Number of pages | 8 |
| DOIs | |
| Publication status | Published - 2018 Jan 1 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1684 |
| ISSN (Print) | 1064-3745 |
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All Science Journal Classification (ASJC) codes
- Molecular Biology
- Genetics
Cite this
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Monitoring changes in the abundance of endogenously expressed ATP-sensitive potassium (KATP) channels in the plasma membrane using surface biotinylation. / Ruan, Jing Syuna; Chen, Pei-chun.
Methods in Molecular Biology. Humana Press Inc., 2018. p. 29-36 (Methods in Molecular Biology; Vol. 1684).Research output: Chapter in Book/Report/Conference proceeding › Chapter
TY - CHAP
T1 - Monitoring changes in the abundance of endogenously expressed ATP-sensitive potassium (KATP) channels in the plasma membrane using surface biotinylation
AU - Ruan, Jing Syuna
AU - Chen, Pei-chun
PY - 2018/1/1
Y1 - 2018/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85032207138&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85032207138&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-7362-0_3
DO - 10.1007/978-1-4939-7362-0_3
M3 - Chapter
C2 - 29058181
AN - SCOPUS:85032207138
T3 - Methods in Molecular Biology
SP - 29
EP - 36
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -