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Active Arf1 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Pierce Active Arf1 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Arf1 GTPase through specific protein interaction with the GGA3 protein-binding domain.

The Active Arf1 Pull-Down and Detection Kit includes purified GST-GGA3 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Arf1 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from MDCK cells, a cell line that is known to have robust Arf1 activity.

Features of the Active Arf1 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Arf1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Arf1 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Arf1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
• Study the activation of Arf1 during the assembly of coat proteins onto budding vesicles or trans-golgi network and endosomes
• Monitor Arf1 activity after stimulation with growth factors
• Monitor Arf1 activity after small molecule inhibitor treatment

The Active Arf1 Pull-Down and Detection Kit was validated for function and specificity of the active Arf1 enrichment method using cell lysates treated with GTPγS to activate endogenous Arf1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Arf1 in the GTP-bound form (active), resulting in a strong signal when endogenous Arf1 is present. GDP treatment pushes Arf1 into the GDP-bound state (inactive), resulting in minimal or no signal, regardless of Arf1 protein levels. The kit is optimized for Western blot detection using an HRP-conjugated secondary antibody (Goat Anti-rabbit IgG, Part No. 31460) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

Arf1 Background:
ADP ribosylation factor proteins 1-6 (Arfs) are members of the Ras family of small GTPases. Although structurally similar, the cellular roles of Arf1-6 are different from the other Arf family members; their endogenous roles are not ADP ribosylation, but rather regulation of heterotrimeric G proteins. The Arf proteins can be divided into three classes: Class I—Arf 1-3; Class II—Arf 4,5; Class III—Arf6. Class I and II Arfs are associated with trans-Golgi network (TGN) and are involved in recruiting effector proteins to the Golgi membrane and forming vesicles. Unlike other GTPases, Arf GTPases are modified by myristoylation at the amino-terminus to allow insertion into the membrane. The Class III protein Arf6 is associated with the plasma membrane and is involved in vesicle formation at the plasma membrane, vesicle recycling and remodeling of the actin cytoskeleton.

Although the Arf GTPases are expressed in all cells, Arf1 is the most abundant, active and best-characterized. Arf1 recruits its effectors, including coatomer and clathrin adaptor complex (AP), to the Golgi for vesicle formation and budding. After recruitment, Arf1 maintains the coat proteins on the membrane until the vesicle is formed. Arf1 then interacts with its effector resulting in the release of Arf1 from the membrane and vesicle budding. The golgi-localized γ-ear-containing Arf (GGA) effector binding proteins contain an amino-terminal VHS domain, a GGA homology domain (GGAH), a proline-rich linker region and a carboxy-terminal γ-ear adaptin homology domain (AGEH). The VHS domain interacts with cytoplasmic domains for receptor sorting, the GGAH domain binds activated Arf, the proline-rich region interacts with clathrin and the AGEH domain interacts with cytosolic proteins. Through mutational studies, the GGAH domain has been shown to be sufficient to bind GTP-bound Arf, and Arf1 binds all three GGA effector proteins. The association of the GGA proteins with the TGN and with the plasma membrane is regulated by the Arf.

More Product Data
Measure activation of small GTPases via their specific downstream effectors

NA-Star™ Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set (Invitrogen™)

The NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set is designed for the rapid and sensitive quantitation of influenza neuraminidase inhibitor resistance in dilutions of virus culture medium in a 96-well microplate format. The NA-Star® Influenza Neuraminidase Inhibitor Reagent kit provides all necessary assay reagents enabling improved global assay standardization and more accurate comparison of results lab-to-lab.

The NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit includes everything you need to quantitate neuraminidase activity and neuraminidase inhibitor resistance in avian, equine, human (types A and B), and porcine influenza viruses. The kit's fast and easy protocol and convenient 96-well plate format make it ideal for monitoring influenza virus neuraminidase inhibitor resistance, as well as high-throughput inhibitor compound screening.

• Includes pre-made solutions for all required reagents, enabling you to standardize assays and compare results between labs easily.
• Provides up to 50-fold higher sensitivity than assays using fluorescent methylumbelliferone N-acetylneuraminic acid (MUNANA) substrate.
• Dynamic range of four orders of magnitude enables you to quantitate virus isolates over a broad range of virus concentration and with varying levels of neuraminidase activity without performing numerous sample dilutions.
• Fast and easy protocol enables you to complete assays in less than 1.5 hours, providing rapid assay throughput.

Get Results in Less than 1.5 Hours
The fast and easy protocol enables you to perform assays in less than 1.5 hours. Simply incubate your virus samples with dilutions of neuraminidase inhibitor, add NA-Star chemiluminescent substrate, incubate again, and then add the accelerator solution, which triggers light emission from the reaction product. Light signal is measured with a luminometer, including multi-mode instruments that include a luminometer mode. For best results, use a luminometer with an automatic injector to add the accelerator solution. You may use a luminometer without an injector, provided you add reagents with a multichannel pipettor and read the plate immediately.

Up to 50-Fold Higher Sensitivity than MUNANA-Based Assays
The chemiluminescent-based detection technology provides a wide dynamic range -- greater than four orders of magnitude of neuraminidase concentration (two orders of magnitude greater than fluorescent MUNANA-based assays) -- enabling you to quantitate neuraminidase inhibitor resistance levels over a broad range of virus concentration and neuraminidase activity without having to test multiple virus dilutions.

Pre-Made Reagents Make it Easy to Standardize Assays
Quality-tested protocols, and pre-made reagents eliminate variability, enabling you to easily standardize assay performance and results across experiments and different laboratories.

Monitor Drug Resistance of Human, Avian, and Livestock Influenza Viruses
The kit's ease of use, standardization, and broad range of compatible species (avian, equine, human types A and B, and porcine influenza viruses) make it an important new tool for assessing and researching drug resistance of influenza in humans, birds, and livestock.

Screen for New Inhibitors and Develop New Vaccines
The kit has many other applications, including screening for new neuraminidase inhibitors and quantitating viral neuraminidase activity for vaccine development. It is also ideal for quantitating neuraminidases from bacteria, including S. typhimurium, C. perfringens, V. cholera, and likely others.

For Research Use Only. Not for use in diagnostic procedures.

Pierce™ Kinase Enrichment Kit with ADP Probe (Thermo Scientific™)

This Thermo Scientific Pierce Kinase Enrichment Kit utilizes an ActivX™ ADP Probe to covalently label the active site of ATPases, including chaperones and metabolic enzymes, to enable their selective enrichment using a desthiobiotin tag.

ActivX ADP Probes feature an amine-reactive nucleotide analog and a desthiobiotin (biotin analog) tag that facilitates selective labeling of lysines in the kinase active site and then subsequent enrichment and recovery of labeled protein. These features allow identification and profiling of target enzyme classes across samples or assessment of the specificity and affinity of enzyme inhibitors.

Features of the Pierce Kinase Enrichment Kit:

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

Applications:
• Profile small-molecule binding affinities and active-site inhibition in a dose-dependent manner
• Identify dozens to hundreds of inhibitor targets and off-targets from tissues, cells and subcellular proteomes
• Enrichment of enzymes based on function

Thermo Scientific ActivX Desthiobiotin-ADP is a nucleotide derivative that covalently modifies the active site of enzymes at conserved lysine residues in the nucleotide binding site. The structure of these probes consists of a modified biotin (desthiobiotin) attached to the nucleotide through a labile acyl-phosphate bond. Desthiobiotin is a biotin analog that binds less tightly to biotin-binding proteins resulting in binding that is easily reversed by biotin displacement, low pH or heat denaturation.

Desthiobiotin-ADP probes can be used to selectively enrich, identify and profile target enzyme classes or assess the specificity of enzyme inhibitors. Because many ATPases and other nucleotide-binding proteins bind nucleotides or inhibitors even when they are enzymatically inactive, the desthiobiotin probes allow profiling of both inactive and active enzymes in a complex sample. Preincubation of samples with small-molecule inhibitors that compete for active sites can be used to determine inhibitor binding affinity. Active-site nucleotide probes also can be used to identify inhibitor off-targets.

These products are subject to a limited use label license.

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ActivX™ Desthiobiotin-ATP Probe
ActivX™ Desthiobiotin-ADP Probe

ActivX™ Desthiobiotin-ADP Probe (Thermo Scientific™)

ActivX Desthiobiotin-ADP Probe, 16 × 9.9µg
Molecular Weight: 994.15
For use with the Pierce Kinase Enrichment kits.

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Pierce™ Kinase Enrichment Kit with ADP Probe

ActivX™ Desthiobiotin-FP Serine Hydrolase Probe (Thermo Scientific™)

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.

Features of the ActivX Desthiobiotin-FP Serine Hydrolase Probe:

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

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.

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

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.

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.

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ActivX™ TAMRA-FP Serine Hydrolase Probe
ActivX™ Azido-FP Serine Hydrolase Probe

Glutathione S-Transferase Fluorescent Activity Kit (Invitrogen™)

The Glutathione S-Transferase Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of glutathione S-transferase activity in serum, plasma, urine and cell lysates.

This complete, ready-to-use kit includes black 96-well plate(s), glutathione S-transferase standard (10 U/mL), glutathione S-transferase substrate, and other components to perform the assay. A 96-well microplate reader capable of reading the fluorescent emission at 460 nm, with excitation at 370-410 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: serum, plasma, urine, and cell lysates
• Sensitivity: 2.70 mU/mL
• Standard curve range: 7.8 mU/mL–500 mU/mL
• Reactivity: species independent

Background
The Glutathione S-Transferase (GST) family of isozymes function to detoxify and neutralize a wide variety of electrophilic molecules by mediating their conjugation with reduced glutathione1. Human GSTs are encoded by 5 gene families, expressed in almost all tissues as four cytosolic and one microsomal forms. Dividing the family by isoelectric points, the basic alpha (pI 8–11), the neutral mu (pI 5–7), and acidic pi (pH<5) classes are populated by additional subclasses, each isozyme displaying differential specificity for given electrophilic molecules. This assay has been validated for human urine, serum, EDTA, heparin plasma, toadfish liver (Opsanus tau), and oyster hemolymph samples. Most cell lysates should also be compatible. GST activity varies across tissues and species, so this kit should measure GST activity from sources other than human.

Assay principle
The Glutathione S-Transferase Fluorescent Activity kit is designed to quantitatively measure the activity of GST present in a variety of samples. A GST standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes a non-fluorescent molecule that is a substrate for the GST enzyme that covalently attaches to glutathione (GSH) to yield a highly fluorescent product. Mixing the sample or standard with the supplied detection reagent and GSH and incubating at room temperature for 30 minutes yields a fluorescent product which is read at 460 nm in a fluorescent plate reader with excitation at 390 nm.

Related links
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Learn more about other immunoassays

NA-Star™ Influenza Neuraminidase Inhibitor Resistance Detection Kit (Invitrogen™)

The NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit is designed for the rapid and sensitive quantitation of influenza neuraminidase inhibitor resistance in dilutions of virus culture medium in a 96-well microplate format. The NA-Star® Influenza Neuraminidase Inhibitor Reagent kit provides all necessary assay reagents enabling improved global assay standardization and more accurate comparison of results lab-to-lab.

The NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit includes everything you need to quantitate neuraminidase activity and neuraminidase inhibitor resistance in avian, equine, human (types A and B), and porcine influenza viruses. The kit's fast and easy protocol and convenient 96-well plate format make it ideal for monitoring influenza virus neuraminidase inhibitor resistance, as well as high-throughput inhibitor compound screening.

• Includes pre-made solutions for all required reagents, enabling you to standardize assays and compare results between labs easily.
• Provides up to 50-fold higher sensitivity than assays using fluorescent methylumbelliferone N-acetylneuraminic acid (MUNANA) substrate.
• Dynamic range of four orders of magnitude enables you to quantitate virus isolates over a broad range of virus concentration and with varying levels of neuraminidase activity without performing numerous sample dilutions.
• Fast and easy protocol enables you to complete assays in less than 1.5 hours, providing rapid assay throughput.

Complete Kit for Measuring the Neuraminidase Inhibitor Resistance of Influenza Viruses
The NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit includes NA-Star chemiluminescent substrate for neuraminidase, all necessary assay reagents, and microplates -- everything you need for the fast, accurate quantitation of neuraminidase inhibitor resistance in influenza virus isolates.

Get Results in Less than 1.5 Hours
The kit's fast and easy protocol enables you to perform assays in less than 1.5 hours. Simply incubate your virus samples with dilutions of neuraminidase inhibitor, add NA-Star chemiluminescent substrate, incubate again, and then add the accelerator solution, which triggers light emission from the reaction product. Light signal is measured with a luminometer, including multi-mode instruments that include a luminometer mode. For best results, use a luminometer with an automatic injector to add the accelerator solution. You may use a luminometer without an injector, provided you add reagents with a multichannel pipettor and read the plate immediately.

Up to 50-Fold Higher Sensitivity than MUNANA-Based Assays
The kit's chemiluminescent-based detection technology provides a wide dynamic range -- greater than four orders of magnitude of neuraminidase concentration (two orders of magnitude greater than fluorescent MUNANA-based assays) -- enabling you to quantitate neuraminidase inhibitor resistance levels over a broad range of virus concentration and neuraminidase activity without having to test multiple virus dilutions.

Pre-Made Reagents Make it Easy to Standardize Assays
Quality-tested protocols, microplates, and pre-made reagents eliminate variability, enabling you to easily standardize assay performance and results across experiments and different laboratories.

Monitor Drug Resistance of Human, Avian, and Livestock Influenza Viruses
The kit's ease of use, standardization, and broad range of compatible species (avian, equine, human types A and B, and porcine influenza viruses) make it an important new tool for assessing and researching drug resistance of influenza in humans, birds, and livestock.

Screen for New Inhibitors and Develop New Vaccines
The kit has many other applications, including screening for new neuraminidase inhibitors and quantitating viral neuraminidase activity for vaccine development. It is also ideal for quantitating neuraminidases from bacteria, including S. typhimurium, C. perfringens, V. cholera, and likely others.

For Research Use Only. Not for use in diagnostic procedures.

Pierce™ GTPase Enrichment Kit with GTP Probe (Thermo Scientific™)

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

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ActivX Desthiobiotin-GTP Probe

Active Rac1 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Active Rac1 Pull-Down and Detection Kit is a complete kit for the selective enrichment and detection of GTP-bound Rac1 GTPase through specific protein interaction with the Pak1 protein-binding domain.

The Active Rac1 Pull-Down and Detection Kit includes purified GST-Pak1 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Rac1 primary antibody, SDS sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH3T3 cells, a cell line that is known to have robust Rac1 activity.

Features of the Active Rac1 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Rac1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Rac1 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Rac1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
• Study Rac1 dependent lamellipodia formation
• Study the role of active Rac1 in cancer and angiogenesis
• Monitor Rac1 activity after stimulation with growth factors
• Screen small molecule inhibitors for their effect on Rac1 activity

The Active Rac1 Pull-Down and Detection Kit was validated for function and specificity of the active Rac1 enrichment method using cell lysates treated with GTPγS to activate endogenous Rac1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Rac1 in the GTP-bound, active form, resulting in a strong signal when endogenous Rac1 is present. GDP treatment pushes Rac1 into the GDP-bound, inactive state, resulting in minimal or no signal, regardless of Rac1 protein levels. The kit is optimized for Western blot detection with an HRP-conjugated secondary antibody (Goat Anti-mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

Rac1 Background:
Rho family GTPases serve many cellular functions, including cell signaling, transcriptional regulation and organization of the actin cytoskeleton. This family of GTPases comprise Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, Rac3, and RhoG), Cdc42 (Cdc42 and G25K), Rnd (Rnd1, Rnd2, and RhoE/Rnd3), RhoBTB family and the Miro family. These GTPases enable signal transduction from the plasma membrane to the cytosol through GPCR, tyrosine kinase, cytokine and adhesion receptors. Attachment to the plasma membrane is accomplished through geranylgeranyl lipid modifications at the carboxy-terminus of the protein. Rac1 activation results in actin polymerization and appears as membrane ruffling at the cellular periphery. Rac1 activation also results in lamelipodia formation.

The Rac1 GTPase transduces signals through tyrosine kinases, adhesion molecules or cytokine/chemokine receptors after stimulation with growth factors (EGF, insulin, PDGF, NGF), integrins (fibronectin) or chemoattractants (fMLP). For example, stimulation with EGF results in PI3 kinase activation, resulting in cell growth and reorganization at the cell periphery (membrane ruffling). Alternatively, tyrosine receptor kinase signaling through Rac1 leads to activation of the MAPK stress response pathways SAPK (JNK) and p38. Stimulation of cells with fibronectin results in integrin-mediated cell spreading. Two of the main effector proteins of Rac1 are Pak1 and phosphoinositol 4-phosphate 5-kinase. Pak1 (p65 Pak) is a kinase that activates the JNK pathway, while phosphoinositol 4-phosphate 5-kinase promotes actin filament assembly. Rac is critical for T-cell development and for promoting differentiating cells. However, the Rho family of GTPases can work agonistically during cell signaling and and antagonistically during differentiation.

More Product Data
Measure activation of small GTPases via their specific downstream effectors
Detection and localization of active GTPases in neuronal cell differentiation

Active Rho Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Pierce Active Rho Pull-Down and Detection Kit enables selective enrichment and detection of GTP-bound Rho GTPase through specific protein interaction with the Rhotekin protein-binding domain.

The Active Rho Pull-Down and Detection Kit includes purified GST-Rhotekin Rho-binding domain (RBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/washing buffer, anti-Rho antibody, secondary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line known to have robust Rho activity.

Features of the Active Rho Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific pan anti-Rho antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Rho detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Rho GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
• Study the activation of Rho during stress fiber formation
• Monitor Rho activity after stimulation with growth factors
• Screen small-molecule inhibitors for their effects on Rho activity

The Active Rho Pull-Down and Detection Kit was validated for function and specificity for active Rho using cell lysates treated with GTPγS to activate endogenous Rho and compared to GDP-treated lysates to inactivate the small GTPase. GTPγS treatment traps Rho in the GTP-bound form (active), resulting in a strong signal when endogenous Rho is present. GDP treatment pushes Rho into the GDP-bound state (inactive), resulting in little to no signal, regardless of Rho protein levels. This kit is optimized for Western blot detection using an HRP-conjugated secondary antibody (included) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (available separately, Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

Kit components can also be used for immunofluorescent staining. Neuronal NS-1 cells were stimulated with NGF to study the spatial distribution of active Rho using the GST-RBD protein and anti-Rho antibody supplied in the kit.

Rho Background:
Rho family GTPases serve many cellular functions, including cell signaling, transcriptional regulation and organization of the actin cytoskeleton. This family of GTPases comprise Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, Rac3, and RhoG), Cdc42 (Cdc42 and G25K), Rnd (Rnd1, Rnd2, and RhoE/Rnd3), RhoBTB family and the Miro family. These GTPases enable signal transduction from the plasma membrane to the cytosol through GPCR, tyrosine kinase, cytokine and adhesion receptors. Attachment to the plasma membrane is accomplished through geranylgeranyl lipid modifications at the carboxy-terminus of the protein. Signal transduction through Rho GTPases results in the reorganization of actin into stress fibers and the formation of focal adhesions. RhoA is localized to the cytosol and plasma membrane, while Rho B is localized to the plasma membrane and membrane vesicles. Like RhoA, RhoC is cytosolic, although it localizes to perinuclear regions.

The Rho GTPases interact with GEF, GAP, GDI and effector proteins for signal transduction. There are over 30 mammalian GEFs for the Rho GTPases, and each GEF contains a DBl homology domain for the nucleotide exchange reaction, a pleckstrin homology domain and additional specific domains for protein-protein interactions. The Rho Gap proteins accelerate the hydrolysis of GTP, and the GDI proteins enable translocation of the Rho GTPases between the cytoplasm and membrane and inhibit the GDP/GTP exchange by Rho GEFs. The GEF and GDI interaction with Rho and the Gα subunit is necessary for signal transduction through GPCRs. Some of the effector proteins include p160 Rho kinase, a serine/threonine kinase involved in stress fiber formation, p140mDia, which triggers reorganization of the actin cytoskeleton, and Rhotekin, a serine/threonine kinase scaffold protein that mediates signaling to activate NF-κB. The Rho family GTPases can work agonistically during cell signaling and and antagonistically during differentiation.

More Product Data
Measure activation of small GTPases via their specific downstream effectors
Detection and localization of active GTPases in neuronal cell differentiation

EnzChek™ Phospholipase A1 Assay Kit (Invitrogen™)

This kits takes our stand-alone assay for phospholipase A1, PLA1 (A10070) and combines the necessary reagents to run 2 to 10 complete 96 well microplate assays that monitor activity in purified enzyme preparations and cell lysates. The importance of phospholipases in cellular signaling, lipid metabolism, inflammatory responses and pathological disorders related to these processes has stimulated demand for fluorescence-based activity monitoring methods. In particular the phospholipases resident in plasma and endothelium can perturb circulating LDL and HDL particles, creating pro-artherogenic forms. Recent evidence is drawing a link between these lipases and the progression of several severe neurodegenerative diseases, including Alzheimer's. Molecular Probes’ fluorogenic phospholipase A1 substrates is designed to provide continuous monitoring of phospholipase A1 (PLA1) in purified enzyme preparations, cell lysates and living cells. PLA-1 improves upon two existing reagents in two ways: first it is now PLA-1 specific and secondly it has improved the assay quality by decreasing initial background noise.

Amplex™ Red Phospholipase D Assay Kit (Invitrogen™)

The Amplex® Red Phospholipase D Assay Kit provides a sensitive and simplemethod to detect Phospholipase D (PLD) activity using a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detect phospholipase D activity levels as low as 10 U/mL
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

PLD activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. PLD converts the phosphatidylcholine (lecithin) to choline, which is then oxidized by choline oxidase to betaine and hydrogen peroxide. In the presence of horseradish peroxidase, huydrogen peroxide reacts with the Amplex® Red reagent in a 1:1 stoichiometric ratio to generate the highly fluorescent product resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

Use Amplex® Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex® UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex® Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex® Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

ActivX™ TAMRA-FP Serine Hydrolase Probe (Thermo Scientific™)

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.

Features of the ActivX TAMRA-FP Serine Hydrolase Probe:

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

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.

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

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.

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.

Related Products
ActivX™ Desthiobiotin-FP Serine Hydrolase Probe
ActivX™ Azido-FP Serine Hydrolase Probe

EnzChek™ Elastase Assay Kit (Invitrogen™)

The EnzChek Elastase Assay Kit provides a sensitive, convenient and fast fluorometric method for measuring elastase or other protease activity in purified enzyme systems, cell/tissue lysates or for screening inhibitors in a high-throughput format. The substrate in the EnzChek kit is our BODIPY-FL-labeled DQ elastin conjugate that is highly labeled so that the fluorescence signal is quenched until enzymatic digestion yields highly fluorescent fragments.

EnzChek™ Myeloperoxidase (MPO) Activity Assay Kit (Invitrogen™)

Myeloperoxidase (MPO) is a unique peroxidase that, in addition to its peroxidation activity, also catalyzes the conversion of hydrogen peroxide (H2O2) and chloride (Cl-) to hypochlorous acid (HOCl). The EnzChek® Myeloperoxidase (MPO) Activity Assay Kit provides assays for the determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates. For detection of chlorination, the kit includes nonfluorescent 3'-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypochlorite (-OCl) to yield fluorescein. Peroxidation is detected using nonfluorescent Amplex® UltraRed reagent (A36006), which is oxidized by the H2O2-generated redox intermediates MPO-I and MPO-II to form a fluorescent product. The EnzChek® Myeloperoxidase Activity Assay Kit can be used to continuously detect these activities at room temperature over a broad dynamic range (1.5 to 200 ng/mL). The speed (30 minutes), sensitivity, and mix-and-read convenience make this kit ideal for measuring MPO activities and for high-throughput screening for MPO-specific inhibitors.