Expression cloning and radiotracer uptakes in Xenopus laevis oocytes

Markovich, D. (2008) Expression cloning and radiotracer uptakes in Xenopus laevis oocytes. Nature Protocols, 3 12: 1975-1980. doi:10.1038/nprot.2008.151


Author Markovich, D.
Title Expression cloning and radiotracer uptakes in Xenopus laevis oocytes
Formatted title
Expression cloning and radiotracer uptakes in Xenopus laevis oocytes
Journal name Nature Protocols   Check publisher's open access policy
ISSN 1750-2799
Publication date 2008-01-01
Sub-type Article (original research)
DOI 10.1038/nprot.2008.151
Open Access Status Not yet assessed
Volume 3
Issue 12
Start page 1975
End page 1980
Total pages 6
Place of publication New York, U.S.
Publisher Nature Publishing Group
Language eng
Subject 1300 Biochemistry, Genetics and Molecular Biology
Abstract This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of proteins. A cDNA library is generated, that is, translated into proteins in Xenopus oocytes, and the function of these proteins is assessed by a radiotracer assay. Their large size, high degree of expression and ease of handling makes Xenopus oocytes an optimal tool for the expression and characterization of protein function when compared with traditional expression systems, such as Escherichia coli, yeast or eukaryotic cell lines. The expected results of this technique include the following: functional identification of novel proteins; molecular (kinetic) characterization of protein function; and determination of functionally relevant residues and domains of membrane proteins, including transporters, ion channels and receptors. The identification of novel proteins can take several months, whereas functional characterization in Xenopus oocytes can take 1 week.
Formatted abstract
This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of proteins. A cDNA library is generated, that is, translated into proteins in Xenopus oocytes, and the function of these proteins is assessed by a radiotracer assay. Their large size, high degree of expression and ease of handling makes Xenopus oocytes an optimal tool for the expression and characterization of protein function when compared with traditional expression systems, such as Escherichia coli, yeast or eukaryotic cell lines. The expected results of this technique include the following: functional identification of novel proteins; molecular (kinetic) characterization of protein function; and determination of functionally relevant residues and domains of membrane proteins, including transporters, ion channels and receptors. The identification of novel proteins can take several months, whereas functional characterization in Xenopus oocytes can take 1 week.
Keyword Biochemistry and protein analysis
Model organisms
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Biomedical Sciences Publications
 
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Created: Thu, 03 Sep 2009, 18:11:23 EST by Mr Andrew Martlew on behalf of Faculty of Science