HUMAN PROTEINPEDIA 


Experimental Details - HuPA_00155
Experiment type GST Pull down
Short description GST Pull down based protein-protein interaction analysis
Experimental description The GST-GTPases RhoA (75 pmol) was stripped of nucleotides by incubating it for 10 min at room temperature in binding buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2.5 ng of bovine serum albumin per ml, 10 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 10% glycerol) supplemented with a cocktail of protease inhibitors (Complete; Roche Molecular Biochemicals). After incubation, the buffer was supplemented with 50 mM MgCl2 plus either GDP (450 μM), GTP (450 μM), or no nucleotides and then incubated 30 min longer. At this moment, 30 μl of glutathione beads (Pharmacia/LKB) of the purified polyhistidine tagged version of human Vav-3 protein, residues 144 to 847, (55 pmol) was added to the GTPase solution. After an incubation of 3 h at 4C, the beads were washed three times with binding buffer plus 50 mM MgCl2, boiled in the presence of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and analyzed by immunoblot using either anti-hexahistidine or anti-GST antibodies.
Principal Investigator's NameDr. Xose Bustelo
TitleProfessor
E-mailxbustelo@usal.es
AddressCenter for Cancer Research
Campus Miguel de Unamuno
Salamanca, Spain 37007
CountrySpain
Lab URLhttp://www.cicancer.org
Data submitted byDr. Nieves Ibarrola
TitlePh.D.
E-mailnibarrola@usal.es
Published/Unpublished Published
Journal name
PubMed ID 10523675
Sample source
Tissue:
Cell line:
Source organism Homo sapiens [Taxonomy:9606]



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