Thermo Scientific™ ActivX™ Desthiobiotin-FP Serine Hydrolase Probe

Description

• ActivX™ FP Probes feature a reactive fluorophosphonate group that specifically and covalently labels the active-site serine of enzymatically active serine hydrolases
• These probes are available with a desthiobiotin (biotin analog) tag for selective enrichment or a reactive- azido group (Staudinger reagents) that facilitate multiplex labeling when used with phosphine- or alkyne-derivatized tags
• ActivX active-site enrichment probes are advantageous for determining active enzyme levels using 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
• Applications of ActivX FP Probes: • Determine serine hydrolase enzyme activity in cells and lysates • Mapping the active-site serine of functionally diverse serine hydrolase family members (e.g
• proteases, lipases, esterases) • Screen for small molecule binding affinities and active-site inhibition • Profile serine hydrolases using fluorescent, Western blot or mass spectrometry workflows The serine hydrolase superfamily is one of the largest, most diverse enzyme families in eukaryotic proteomes
• Serine hydrolases are generally grouped into two large families: serine proteases (e.g., trypsin, elastase and thrombin) and metabolic serine hydrolases
• Metabolic serine hydrolases are divided into multiple enzyme subclasses (e.g., esterases, lipases, amidases and peptidases) based on structure, catalytic mechanism and substrate preference
• These probes can be used to assess activity or screen small molecule inhibitors against enzymes derived from cell lysates, subcellular fractions, tissues and recombinant proteins
• 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
• The Azido-FP probe can be used in combination with a phosphine- or alkyne-derivatized tag for either detection or enrichment of serine hydrolases using Western blot or mass spectrometry
• These products are subject to a limited use label license.