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GTPase Enrichment Kit and Probes


Selective capture and enrichment of GTPases using active-site probes.

Thermo Scientific Pierce GTPase Enrichment Kit with ActivX GTP Probes for GTPases.

Thermo Scientific Pierce GTPase Enrichment Kits use ActivX™ GTP Probes to covalently label the active site of GTPases and the GTPase subunits of G-protein coupled receptors enabling their selective enrichment using the desthiobiotin tag.

ActivX™ GTP Probes feature a desthiobiotin (biotin analog) tag that facilitates selective enrichment and recovery of labeled GTPases allowing identification and profiling of target enzyme classes across samples or to assess the specificity and affinity of enzyme inhibitors.

 

Highlights:

  • Specific – label only the conserved active-site lysines of nucleotide-binding proteins
  • Compatible – use for in vitro labeling of GTPase enzymes derived from cells or tissues.
  • Flexible – use with Western blot or mass spectrometry (MS) workflows

Applications:

  • Broad enrichment of GTP-binding proteins from tissues, cells and subcellular proteomes
  • Enrichment of enzymes based on function
  • Profiling of dozens to hundreds of inhibitor targets

Product Details:

Pierce GTPase Enrichment Kits with ActivX GTP Probes enable selective labeling and enrichment of small GTPases and large G-protein subunits. The ActivX Desthiobiotin-GTP Probe structure consists of a modified biotin attached to the nucleotide by a labile acyl-phosphate bond. 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.

GTPases identified by mass spectrometry is ActivX GTP Probes. Number for GTPase family members from human cell lysates identified by mass spectrometry after labeling and enrichment using the desthiobiotin-GTP probe. Data provided by ActivX Biosciences, Inc.
GTPase family Number identified
Rab 38
Ras 9
Arf 8
Rho 5
4
Sar1 2

Assessment of active-site labeling 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.

A. Reaction overview for active site probes and nucleotide binding binding sites in ATPases or other kinases
B.
Mechanism and chemical structures of Thermo Scientific Active Site Probes for GTPases. A. Nucleotide analogues bind to the active sites of GTPases and the biotin affinity tag is irreversibly transferred to highly conserved lysine residues in the active site. B. Desthiobiotin is attached to the GTP nucleotide through a labile acyl phosphate linkage, allowing efficient desthiobiotin label transfer to amines near the active site of GTPases. Desthiobiotin binding to streptavidin is easily reversible under acidic elution conditions, allowing high recovery of labeled proteins and peptides.

Workflow for targeted capture and analysis of kinases, GTPases and serine hydrolases using Thermo Scientific Pierce Active Site Probes.
Assessment of active-site labeling can be accomplished by Western blot or mass spectrometry (MS). For the Western blot workflow, desthiobiotin-labeled proteins are enriched, analyzed by SDS-PAGE and detected with specific antibodies. For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested. Only the desthiobiotin-labeled, active site peptides are enriched for LC-MS/MS analysis. Both workflows can be used to determine inhibitor target binding, but the MS workflow also can identify global inhibitor targets and off-targets and provide higher throughput for quantitative assays.

RAS CDC42 and Rho A labeled and detected by Western blotting using ActivX GTPase probes.

RAC1 labeled and detected by Western blot using ActivX GTPase probes.

Labeling and Enrichment of small GTPases with desthiobiotin-GTP probe using the Western blot workflow. A. A549 cell lysates (500µg) were treated with (+) or without (-) 20mM magnesium chloride after labeling with 20µM of desthiobiotin-GTP probe. Desthiobiotin-labeled proteins were denatured and enriched using streptavidin agarose before separation by SDS-PAGE and Western blotting with specific GTPase antibodies. B. Recombinant RAC1 was pretreated with GTPγS before labeling with desthiobiton-GTP in the presence (+) or absence (-) of 20mM magnesium. Samples were separated by SDS-PAGE and analyzed Western blot (Labeled RAC1) to detect biotinylation of the active site. GelCode Blue stain (Total RAC1) was used to stain a duplicate gel to show equal loading.

References:

  1. Patricelli, M.P., et al. (2007). Functional Interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46:350-358.
  2. Cravatt, B.F., Wright, A.T., and Kozarich, J.W. (2008). Activity-based protein profiling: From enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77:383–414.
  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.

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

Related Products:

Pierce Kinase Enrichment Kits
Active GTPase Pull-Down and Detection Kits
Browse Products for Mass Spectrometry
Browse Products for Western blotting
Find Secondary Detection Reagents for blotting and enrichment
Halt Phosphatase Inhibitor Cocktails


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
88314 Pierce GTPase Enrichment Kit with GTP Probe
Formulation: Multi-component kit
Sufficient For: 16 pull-down assays

Kit Contents:
ActivX Desthiobiotin-GTP Probe, 16 x 12.9µg
Pierce IP Lysis Buffer, 100mL
Reaction Buffer, 125mL
Halt Protease/Phosphatase Inhibitor Cocktail (100X), 1mL
Zeba Spin Desalting Columns, 7K MWCO, 8 x 5mL columns
High Capacity Streptavidin Agarose Resin (50% slurry), 1mL
MgCl2, 500µL
Urea, 12g

Storage: ActivX Desthiobiotin-GTP Probe is shipped separately with dry ice and stored at -80°C upon receipt. All other kit components are shipped at ambient temperature and stored at 4°C upon receipt.
16-rxn Kit Product Instructions for product #88314 Pierce GTPase Enrichment Kit with GTP Probe MSDS for product #88314 Pierce GTPase Enrichment Kit with GTP Probe $450.00

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88315 ActivX Desthiobiotin-GTP Probe
Formulation: Desthiobiotin-GTP, MW 1275
16 x 12.9µg Product Instructions for product #88315 ActivX Desthiobiotin-GTP Probe MSDS for product #88315 ActivX Desthiobiotin-GTP Probe Certificate of Analysis for product #88315 ActivX Desthiobiotin-GTP Probe $225.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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