Thermo Scientific™ ActivX™ Desthiobiotin-GTP Probe

Description

• ActivX™ Desthiobiotin-GTP Probes contain a modified biotin attached to the nucleotide by a labile acyl-phosphate bond
• ActivX active-site enrichment probes are advantageous for determining active enzyme levels through the use of analog-tagged probes to allow profiling of both inactive and active enzymes
• The ActivX probes selectively enrich only those enzymes that are functionally active and biologically relevant at the time of labeling
• With selective enrichment, one can identify and profile target enzyme classes across samples or can assess the specificity and affinity of enzyme inhibitors
• After removal of GTP or GDP nucleotides from enzymes, the desthiobiotin-GTP probe can be used to covalently modify conserved lysine residues in the GTPase nucleotide-binding site
• Desthiobiotin-GTP can selectively enrich, identify and profile target enzyme classes in samples
• Pre-incubation of samples with small-molecule inhibitors that compete with active-site probes can be used to determine inhibitor binding affinity and target specificity
• The ActivX Desthiobiotin-GTP Probe covalently labels the active site of GTPases and GTPase subunits of G-protein coupled receptors
• Applications of ActivX Desthiobiotin-GTP Probe: • Broad enrichment of GTP-binding proteins from tissues, cells and sub-cellular proteomes • Enrichment of enzymes based on function • Profiling of dozens to hundreds of inhibitor targets Assessment of active-site labeling with desthiobioin probes can be accomplished by either Western blot or mass spectrometry (MS)
• For the Western blot workflow, desthiobiotin-labeled proteins are enriched for SDS-PAGE analysis and subsequent detection with specific antibodies
• For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested to peptides
• Only the desthiobiotin-labeled, active-site peptides are enriched for analysis by LC-MS/MS
• Both workflows can be used for determining inhibitor target binding, but only the MS workflow can identify global inhibitor targets and off-targets
• These products are subject to a limited use label license.