Pierce™ GTPase Enrichment Kit with GTP Probe

Catalog number: 88314

Thermo Scientific™  Related applications: Protein Assays and Analysis | Protein Mass Spectrometry Analysis | Protein Purification & Isolation

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Description

Thermo Scientific Pierce GTPase Enrichment Kit with GTP probe uses ActivX™ GTP Probes to covalently label the active site of GTPases and GTPase subunits of G-protein coupled receptors enabling their selective enrichment using the desthiobiotin tag allowing identification and profiling of target enzyme classes across samples or to assess the specificity and affinity of enzyme inhibitors.

Features of GTPase Enrichment Kit with GTP Probe:

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 of GTPase Enrichment Kit with 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

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.

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.

More Product Data
GTPase enrichment using a new active-site probe

Related Products
ActivX Desthiobiotin-GTP Probe
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Format: Tube(s), Spin Column
Technique: Pull-Down
Assay Type: Protein Quantitation Assay
Product Size: 16 reactions
Target Enzyme: GTPases, Phosphodiesterases, Kinases (Multiple), Phosphatases, ATPases
Substrate Type: GTPase Substrate
Detection Method: Chemiluminescent, Mass Spectrometry, Western Blot
Substrate Properties: Chemical Substrate

Contents & storage

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

ActivX Desthiobiotin-GTP Probe is shipped separately with dry ice and should be stored at -80°C. All other kit components are shipped at ambient temperature and stored at 4 deg;C.

Documents

Manuals & protocols