Update profile
    Change password
    Sign out

Active Serine Hydrolase Probes


Probes for specific detection and enrichment of active serine hydrolases.

Thermo Scientific Pierce Kinase Enrichment Kit with ActivX ATP or ADP Probes for ATPases.

Thermo Scientific Serine Hydrolase Probes are ActivX™ Fluorophosphonate (FP) and other tagged phosphonate probes to purify or assay serine hydrolase active sites using fluorescence, biotin-affinity, or mass spectrometry.

These 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, TAMRA fluorophore for detection or a reactive- azido group (Staudinger reagents) that facilitates multiplex labeling when used with phosphine- or alkyne-derivatized tags. 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.

Highlights:

  • Specific – labels the reactive site of active serine hydrolases
  • Compatible – tags available for capture, detection and Staudinger conjugation
  • Flexible – use for in vitro or intracellular enzyme labeling

Applications:

  • 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

Product Details:

ActivX active-site probes are especially advantageous for determining active enzyme levels compared to other protein expression profiling techniques that only measure abundance. Because many of the proteolytic enzymes in the serine hydrolase family are expressed as inactive proenzymes (zymogens), the ActivX FP probes selective enrichment only those enzymes that are functionally active and biologically relevant at the time of labeling. This feature also makes it possible to perform selective screening of inhibitors or other conditions that alter enzyme activity.

Serine hydrolases identified by mass spectrometry with ActivX FP Probes. Number of serine hydrolase family members from mouse brain and liver tissue extracts identified by mass spectrometry after labeling and enrichment using the desthiobiotin-FP probe.
Serine hydrolase family Number identified
Hydrolases 10
Esterases 6
Lipases 5
Peptidases 4
Other 4

Active serine hydrolases labeled with ActivX FP Probes can be detected and quantified by Western blot, fluorescent gel imaging or mass spectrometry by using a compatible tag. The TAMRA-FP probe can be used to label and detect active serine hydrolases in samples by fluorescent gel imaging, capillary electrophoresis or mass spectrometry. An anti-TAMRA antibody is also available for immuno-enrichment of TAMRA-FP probe-labeled proteins. The Azido-FP probe is used in combination with a phosphine- or alkyne-derivatized tag for either detection or enrichment. Desthiobiotin-FP probes can be used for streptavidin-based enrichment and detection of active serine hydrolase proteins in Western blotting or mass spectrometry.

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.

A. Reaction overview for activx fluorophosphonate probes for serine hydrolase active sites
B. Structures of azid-fp,  desthiobiotin-fp and TAMRA-fp serine hydrolase probes
Mechanism and chemical structures of Thermo Scientific Active Site Probes for serine hydrolases. A. Fluorophosphonate probes covalently and specifically attach to the active site serine of active serine hydrolases and proteases. B. Structures of the azido, desthiobiotin and fluorescent tagged fluorophosphonate probes for labeling, affinity enrichment or fluorescent detection of active serine hydrolase enzymes.

workflow for the serine hydrolase active site probes using fluorophosphonate detection method workflows for serine hydrolase probes from ActivX
Workflows for detection, targeted capture and analysis of serine hydrolase enzymes using Serine Hydrolase Probes. Protein and peptide workflows for the profiling, capture, and detection of serine hydrolases with ActivX FP Probes. Preincubation of enzymes with inhibitors allows for the determination of inhibitor specificity, binding affinity and potency by Western blot (or fluorescent SDS-PAGE) of probe-labeled proteins or mass spectrometry of probe-labeled peptides.

inhibitor screening for serine hydrolase in mouse liver and brain.
Screening of different inhibitors in mouse tissue lysates using serine hydrolase probes. A. Mouse brain or liver tissue lysates (50µg) were pretreated with either DMSO (o) or serine hydrolase inhibitors 100µM AEBSF (a), URB597 (u) or CAY10401 (c) for 1 hour before labeling with 2µM TAMRA-FP probe. Labeled proteins were separated by SDS-PAGE and analyzed by fluorescent gel scanning using a GE Healthcare Typhoon™ Imager. Unlabeled lysate (–) and heat denatured (Δ) lysate were used as a controls to show probe labeling specificity. FAAH is indicated by double arrow. B. Mouse brain tissue lysate (500µg) was pretreated as in panel A and labeled with 2µM desthiobiotin-FP probe. Desthiobiotin-FP labeled proteins were denatured and enriched using streptavidin agarose before separation by SDS-PAGE and Western blotting with a specific FAAH antibody.

active serine mapping for FAAH using acitvX dethiobiotin-fp probe.
Identification of the active-site serine of FAAH.The peptide containing the desthiobiotin-FP modified active-site serine (lower case "s") of FAAH was identified on a Thermo Scientific LTQ Orbitrap XL mass spectrometer using IT ETD MS/MS fragmentation.

References:

  1. Liu,Y., et al. (1999). Activity-based protein profiling: The serine hydrolases. Proc Natl Acad Sci USA 96(26):14694-9.
  2. Patricelli, M.P., et al. (2001). Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 1:1067-71.
  3. Okerberg, E.S., et al. (2005). High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci USA 102(14):4996-5001.
  4. Simon, G.M. and Cravatt, B.F. (2010). Activity-based proteomics of enzyme superfamilies: Serine hydrolases as a case study. JBC 285(15):11051-5
  5. Okerberg, et al. (2005). High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci USA. 102(14):4996-5001.

Related Resources:

Activity-Based Protein Profiling Technology Licensed from ActivX Biosciences, Inc. (opens pdf)
ActivX KiNativ™ in situ Kinase Profiling Services (opens new window)
Review of sample preparation for mass spectrometry
Mass Spectrometry Sample Prep Workflow
Desthiobiotin product selection guide

Related Products:

Anti-TAMRA Monoclonal Antibody (order below or follow link to view on Pierce-Antibodies.com)
GTPase Enrichment Kits and Probes
Kinase Enrichment Kits and Probes
Browse Products for Mass Spectrometry
Browse Products for Western blotting
Find Secondary Detection Reagents


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
88316 ActivX Azido-FP Serine Hydrolase Probe
Formulation: Azido-fluorophosphonate, MW 350
Sufficient For: 100 labeling reactions or 10 pull-down assays
3.5µg Product Instructions for product #88316 ActivX Azido-FP Serine Hydrolase Probe MSDS for product #88316 ActivX Azido-FP Serine Hydrolase Probe Certificate of Analysis for product #88316 ActivX Azido-FP Serine Hydrolase Probe $325.00

Add to cart
88317 ActivX Desthiobiotin-FP Serine Hydrolase Probe
Formulation: Desthiobiotin-fluorophosphonate, MW 464
Sufficient For: 100 labeling reactions or 10 pull-down assays
4.6µg Product Instructions for product #88317 ActivX Desthiobiotin-FP Serine Hydrolase Probe MSDS for product #88317 ActivX Desthiobiotin-FP Serine Hydrolase Probe Certificate of Analysis for product #88317 ActivX Desthiobiotin-FP Serine Hydrolase Probe $325.00

Add to cart
88318 ActivX TAMRA-FP Serine Hydrolase Probe
Formulation: TAMRA-fluorophosphonate, MW 680
Sufficient For: 100 labeling reactions or 10 pull-down assays
6.8µg Product Instructions for product #88318 ActivX TAMRA-FP Serine Hydrolase Probe MSDS for product #88318 ActivX TAMRA-FP Serine Hydrolase Probe Certificate of Analysis for product #88318 ActivX TAMRA-FP Serine Hydrolase Probe $325.00

Add to cart
MA1041 Anti-TAMRA Monoclonal Antibody (5G5)
100µg MSDS for product #MA1041 Anti-TAMRA Monoclonal Antibody (5G5) $316.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

Recommend this page

Follow Us
Email Sign-up  Email Announcements

You may also need
34065 - Metal Enhanced DAB Substrate Kit

 



Antibodies  |   Molecular Biology   |   Cell Biology   |  Thermo Scientific  |   * Trademarks  |   Privacy Statement
© 2014 Thermo Fisher Scientific Inc.   |   3747 N Meridian Rd, Rockford, IL USA 61101   |   1-800-874-3723  or  815-968-0747