Linked Life Data

16289967

Source:http://linkedlifedata.com/resource/pubmed/id/16289967

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2005-11-18
pubmed:abstractText
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 (32)Pi 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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1046-2023
pubmed:author
pubmed:issnType
Print
pubmed:volume
37
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
190-6
pubmed:dateRevised
2011-5-5
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Measuring Ras-family GTP levels in vivo--running hot and cold.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Walther Cancer Institute, Indianapolis, IN 46202, USA.
pubmed:publicationType
Journal Article