Measuring Ras-family GTP levels in vivo - Running hot and cold

Ariel F. Castro, John F. Rebhun, Lawrence Quilliam

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The detection of Ras-family GTPase activity is important in the determination of cell signaling events elicited by numerous ligands and cellular processes. This has been made much easier in recent years by the use of glutathione S-transferase (GST)-fused Ras binding domains. These domains from downstream effectors such as Raf and RalGDS preferentially bind the GTP-bound Ras proteins enabling their extraction and subsequent quantification by immunoblotting. Despite this advance, effectors that efficiently discriminate between GTP- and GDP-bound states are not available for many Ras-family members. While this hampers the ability to detect activity in tissue specimens, it is still possible to metabolically label cells with 32Pi to load the GTP/GDP pool with labeled nucleotides, immunoprecipitate the Ras protein and detect the bound label following thin layer chromatographic separation and exposure to film or a phosphorimager. Using a transfection system and antibodies that recognize epitope tags one can test the ability of a protein to work as a GEF or GAP for a certain GTPase. Alternatively, if an immunoprecipitating antibody is available to the target GTPase, then analysis of endogenous GTP/GDP ratio is possible. Here we describe the detection of M-Ras and Rap1 activity by GST-RBD pull-down as well as that of Rheb and epitope-tagged R-Ras by classical metabolic labeling and immunoprecipitation.

Original languageEnglish
Pages (from-to)190-196
Number of pages7
JournalMethods
Volume37
Issue number2
DOIs
StatePublished - Oct 2005

Fingerprint

Guanosine Triphosphate
ras Proteins
GTP Phosphohydrolases
Aptitude
Glutathione Transferase
Labels
Epitopes
Cell signaling
Antibodies
Immunoprecipitation
Immunoblotting
Labeling
Transfection
Nucleotides
Tissue
Ligands
Proteins

Keywords

  • GTP
  • GTPase
  • Guanine nucleotide exchange
  • M-Ras
  • R-Ras
  • Rap1
  • Ras
  • Ras binding domain
  • Rheb

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Measuring Ras-family GTP levels in vivo - Running hot and cold. / Castro, Ariel F.; Rebhun, John F.; Quilliam, Lawrence.

In: Methods, Vol. 37, No. 2, 10.2005, p. 190-196.

Research output: Contribution to journalArticle

Castro, Ariel F. ; Rebhun, John F. ; Quilliam, Lawrence. / Measuring Ras-family GTP levels in vivo - Running hot and cold. In: Methods. 2005 ; Vol. 37, No. 2. pp. 190-196.
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