An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay

Keiko Kadono-Okuda, Douglas A. Andres

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [α- 32P]GTP. The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E. coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-like small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This results in the isolation of predominantly full- length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particularly useful for the cloning of novel Ras-related GTP- binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

Original languageEnglish
Pages (from-to)187-191
Number of pages5
JournalAnalytical Biochemistry
Issue number2
StatePublished - Dec 15 1997

Bibliographical note

Funding Information:
1This work was supported by National Institutes of Health Grant EY 11231.

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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