Thermo Scientific™ Sonde con fluorofosfonati ActivX™

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.