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PKA (Protein Kinase A) Colorimetric Activity Kit (Invitrogen™)

The PKA Activity research-use-only kit is a colorimetric activity assay designed for the quantification and detection of protein kinase A activity in cell lysates, tissue extracts and buffer samples.

This complete, ready-to-use kit includes a PKA substrate-coated 96-well plate(s), PKA standard (5,000 U), phospho-PKA substrate antibody, and other components to perform the assay. A 96-well microplate reader capable of reading optical density at 450 and 650 nm is required for use of this kit.

Performance characteristics
• Assay type: colorimetric activity kit
• Sample types: cell lysates, tissue extracts, buffer samples
• Sensitivity: 0.366 U/mL
• Standard curve range: 5 U/mL–25 U/mL
• Reactivity: species independent

Background
PKA is a member of an important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, that includes cAMP-dependent protein kinase (PKA or cAPK), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position 3 relative to the phosphorylated serine or threonine. The second messenger cyclic AMP (cAMP) activates PKA in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation2. Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. PKA shares substrate specificity with Akt (PKB) and PKC. Substrates that present this consensus sequence and are phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3.

PKA has been implicated in numerous cellular processes, including modulation of other protein kinases, regulation of intracellular calcium concentration, and regulation of transcription. Transcriptional responses to increased cAMP occur through activation of the cAMP response element–binding protein (CREB), cAMP response element modulator (CREM), and activating transcription factor 1 (ATF1). Each of these transcription factors contains a kinase-inducible domain containing a conserved site for phosphorylation by PKA.

Assay principle
The PKA activity kit is designed to quantitatively measure PKA activity in a variety of samples. A recombinant PKA standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes an immobilized PKA substrate bound to a microtiter plate. Samples containing PKA will, in the presence of the supplied ATP, phosphorylate the immobilized PKA substrate. After a 90-minute incubation followed by a wash, a rabbit antibody specific for the phospho-PKA substrate binds to the modified immobilized substrate. An antibody specific for rabbit IgG labeled with peroxidase is also added to the plate to bind to the rabbit anti-phospho-PKA substrate. After a short incubation and wash, substrate is added and the intensity of the color developed is directly proportional to the amount of PKA in the samples and standards.

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NA-Fluor™ Influenza Neuraminidase Assay Kit (Invitrogen™)

The NA-Fluor™ Influenza Neuraminidase Assay Kit provides validated reagents and a standardized protocol for conducting neuraminidase enzyme assays, including neuraminidase inhibitor (NI) susceptibility screening using a fluorescent MUNANA substrate.

Key product features:
• Efficient design—neuraminidase assay in one complete kit
• Optimized formulations—assay reagents have been optimized for maximum performance based on NISN protocols
• Robust application—functional detection of NI-resistant virus in mixed viral populations
• Easy of use & flexible screening—stable fluorescent signal enables batch-mode or high throughput processing

Neuraminidase Assasy in One Complete Kit
The NA-Fluor™ Influenza Neuraminidase Assay Kit includes comprehensive protocols for titering viral isolates based upon neuraminidase activity and conducting neuraminidase enzyme inhibition assays. The assay can also be used for monitoring neuraminidase enzyme activity from non-viral or bacterial sources. The NA-Fluor™ Kit contains reagents for 10 96-well microplates sufficient for a total of 960 assay wells and includes the following components: fluorescent MUNANA neuraminidase substrate, assay buffer, and stop solution to enhance and stabilize signal. The assay is performed in standard black microplates (microplates not provided in kit) and is read on standard fluorometers.

Neuraminidase Assay Compares to NISN Protocols
The NA-Fluor™ assay reagent formulations are optimized to be comparable to several well-established MUNANA-based assay protocols, such as:
• The MUNANA substrate concentration, assay buffer formulation and assay conditions are consistent with NISN IC-50 determination protocols
• Data generated using the NA-Fluor™ assay corresponds to data generated with established MUNANA based protocols
These key parameters enable investigators to compare data acquired during current NI resistance surveillance screens using the NA-Fluor™ assay to data acquired using their previous MUNANA assay protocols (Figure 1).

Detection of NI-Resistant Virus in Mixed Viral Populations
The large shift in IC-50 values between sensitive and oseltamivir-resistant virus using the NA-Fluor™ assay enables detection of mutant virus in mixed viral samples (Figure 2). This capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during NI susceptibility surveillance.

Flexibility for Screening Neuraminidase Activity
The NA-Fluor™ assay provides an easy, flexible format for the screening of several to several hundred viral isolates, or the screening of thousands of compounds during high-throughput lead discovery with quality data at high confidence levels. With superior performance, the assay has demonstrated a Z´ of 0.78-0.8, making it strongly capable for use in high-throughput screening. The fluorescent reaction product remains stable for hours at room temperature after the assay is complete, enabling read-time flexibility and comparable data from first plate to last. The assay signal remains nearly constant and IC-50 values (data not shown) are identical from data collected up to 4 hours at room temperature and up to 4 days at 4 °C after assay termination (Figure 3). The NA-Fluor™ assay has been optimized as an end-point assay run at 37 °C for one hour following NI pre-incubation. However, the rate of MUNANA substrate turnover remains linear for more than 2 hours with viral neuraminidase, allowing the assay to be performed for as little as 20 minutes to save time or for as long as 2 hours to increase signal output. The assay can also be conducted in real-time without the addition of stop solution for those investigators who want to perform their own assay development or monitor rates of substrate turnover in the presence of inhibitor (Figure 4).

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

Acetylcholinesterase Fluorescent Activity Kit (Invitrogen™)

The Acetylcholinesterase Fluorescent Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of acetylcholinesterase activity in serum, plasma, and erythrocyte membrane samples.

This complete, ready-to-use kit includes black 96-well plate(s), acetylcholinesterase standard (1,000 mU/mL), acetylcholinesterase substrate, acetylthiocholine iodide, and other components to perform the assay. A fluorescence 96-well microplate reader capable of reading fluorescent emission at 510 nm, with excitation at 390 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: serum, plasma, and erythrocyte membrane samples
• Sensitivity: 0.218 mU/mL
• Standard curve range: 6.25 mU/mL–100 mU/mL
• Reactivity: species independent

Background
Acetylcholine (ACh) is an essential neurotransmitter in the central and peripheral nervous systems. In the brain multiple areas exist where cholinergic neurons are concentrated. Nicotinic and muscarinic ACh receptors are recognized as binding and effector proteins to mediate chemical neurotransmission at neurons, ganglia, heart, and smooth muscle fibers and glands.

Acetylcholinesterase is encoded by the single AChE gene; the structural diversity in the gene products arises from alternative mRNA splicing and post translational associations of catalytic and structural subunits. The major form of acetylcholinesterase found in brain, muscle, and other tissues is the hydrophilic species, which forms disulfide-linked oligomers with collagenous, lipid-containing structural subunits. The other alternatively-spliced form, expressed primarily in the erythroid tissues, is structurally different at the C-terminal end and contains a cleavable peptide with a GPI anchor. It associates with membrane receptors through phosphoinositide moieties added post-translationally. This assay has been validated for serum, EDTA and heparin plasma, and solubilized RBC ghosts from a variety of species.

Assay principle
The Acetylcholinesterase Activity kit is designed to quantitatively measure acetylcholinesterase activity in a variety of samples. A human AChE 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 proprietary non-fluorescent molecule, that covalently binds to the thiol product of the reaction between the AChE Substrate and AChE in the standards or samples, yielding a fluorescent product read at 510 nm in a fluorescent plate reader with excitation at 390 nm. The kit is suitable for measuring AchE activity in appropriately diluted serum, plasma, and RBC ghosts from a number of species. It will also measure AChE in extracted tissue samples and cell lysates.

AChE unit definition
One unit will hydrolyze 1.0 μmol of acetylthiocholine iodide per minute at pH 7.4 and 37˚C.

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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.

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Measure activation of small GTPases via their specific downstream effectors

KDalert™ GAPDH Assay Kit (Invitrogen™)

The Ambion® KDalert™ GAPDH Assay Kit is for the reliable measure of GAPDH enzyme activity in cultured human, mouse, or rat cells in less than 30 minutes using a microplate fluorometer. The kit includes sufficient reagents for 375 reactions.

• Assess GAPDH siRNA delivery in 1/3 the time for 1/3 the cost of real-time PCR
• Analyze 1-96 samples simultaneously
• Measure both GAPDH siRNA-induced knockdown AND transfection-induced toxicity
• Compatible with a wide variety of cells and a broad range of culture conditions

The KDalert GAPDH Assay Kit is an ideal positive control for transfection optimization experiments and also measures transfection induced cytoxicity. It is designed for use with Ambion® Silencer® GAPDH siRNA.

Rapid, Time-Saving Procedure
Use the assay to optimize siRNA transfection by transfecting individual cell samples with a GAPDH siRNA and a negative control siRNA. Two to three days after transfection, simply add the included cell lysis buffer to the cells, incubate for 20 minutes, add the diluted master mix of assay reagents, and read the increase in fluorescence four minutes later using a microplate or standard fluorometer. The assay procedure can be completed in about 30 minutes with minimal sample handling.

One Assay for Two Readouts
Because GAPDH is expressed at relatively constant levels, the assay can also be used to monitor transfection agent induced toxicity. For this analysis, GAPDH enzyme activity from negative control siRNA-transfected cells is compared to that of untreated cells. Reduced GAPDH activity in negative control-transfected cells compared to non-transfected cells is an indication that the transfection-induced cytotoxicity.

Accessory Products:
The KDalert™ Kit is designed for use with Silencer® GAPDH siRNAs (SKUs #AM4605, AM4633, AM4634, AM4624, AM4632, or AM4631). Additional KDalert™ Lysis Buffer (SKU #AM8790G) is also available separately.

Butyrylcholinesterase Fluorescent Activity Kit (Invitrogen™)

The Butyrylcholinesterase Fluorescent Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of butyrylcholinesterase activity in serum and plasma samples.

This complete, ready-to-use kit includes black 96-well plate(s), butyrylcholinesterase standard (200 mU/mL), butyrylcholinesterase substrate, and other components to perform the assay. A fluorescence 96-well microplate reader capable of reading fluorescent emission at 510 nm, with excitation at 390 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: serum and plasma samples
• Sensitivity: 0.018 mU/mL
• Standard curve range: 0.3 mU/mL–20 mU/mL
• Reactivity: species independent

Background
Butyrylcholinesterase (BChE) belongs to the same structural class of proteins as acetylcholinesterase (AChE). The 440 kDa tetrameric glycoprotein is predominantly found in blood, kidneys, intestine, liver, lung, heart, and the central nervous system. Many species, such as human, horse, and mice exhibit high BChE activity in plasma, whereas rats have higher acetylcholinesterase activity in plasma1. BChE preferentially acts on butyrylcholine, but also hydrolyzes acetylcholine. This assay has been validated for serum, EDTA and heparin plasmas from a variety of species.
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Assay principle
The Butyrylcholinesterase Activity kit is designed to quantitatively measure butyrylcholinesterase (BChE) activity in a variety of samples. A human BChE 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 proprietary non-fluorescent detection reagent that covalently binds to the thiol product of the reaction between the BChE Substrate and BChE in the standards or samples, yielding a fluorescent product read at 510 nm in a fluorescent plate reader with excitation at 390 nm. The kit is suitable for measuring BChE activity in appropriately diluted serum or plasma from a number of species. It will also measure BChE in extracted tissue samples, CSF, and cell lysates.

BChE unit definition
One unit will hydrolyze 1.0 μmol of butyrylcholine to choline and butyrate per minute at pH 8.0 and 37˚C.

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Active Cdc42 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Active Cdc42 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Cdc42 GTPase through specific protein interaction with the Pak1 protein-binding domain.

The Active Cdc42 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-Cdc42 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line that is known to have robust Cdc42 activity.

Features of the Active Cdc42 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Cdc42 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Cdc42 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Cdc42 GTPase during cell differentiation, migration, division, and cytoskeletal rearrangement
• Study the activation of Cdc42 during filopodia formation
• Monitor Cdc42 activity after stimulation with growth factors
• Screen small molecule inhibitors for their effects on Cdc42 activity

The Active Cdc42 Pull-Down and Detection Kit was validated for the function and specificity of the active Cdc42 enrichment method using cell lysates treated with GTPγS to activate endogenous Cdc42 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Cdc42 in the GTP-bound, active form, resulting in a strong signal when endogenous Cdc42 is present. GDP treatment pushes Cdc42 into the GDP-bound, inactive state, resulting in minimal or no signal regardless of Cdc42 protein levels. This 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.

Cdc42 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. Cdc42 activation results in the polymerization of actin filaments and filopodia formation.

The Cdc42 subfamily of RhoGTPase has been less well-characterized than the Rho and Rac families. Both Cdc42 and Rac1 are activated through tyrosine receptor kinase signaling leading to SAPK and p38 stress kinase pathway activation. Cdc42 is also activated by the chemoattractant fMLP in neutrophils. Fibronectin activates Cdc42 and Rac1 to induce cell spreading, and stimulation with TNF-alpha and IL-1 results in changes in the actin cytoskeleton. There is significant cross-talk between Cdc42 and Rac1, as they act in overlapping pathways, and in some cases, Cdc42 may act upstream of Rac1 during signal transduction. Some of the main effector proteins of Cdc42 are Pak1, N-WASP and IQGAP. Pak1 is a kinase involved in the activation of JNK in the SAPK stress pathway. N-WASP is an effector that induces filopodia formation, and IQGAP interacts with F-actin filaments. In differentiating neurons, Cdc42 plays an active role in neurite outgrowth. However, the Rho family of GTPases can work agonistically during cell signaling and antagonistically during differentiation.

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P450 Demethylation Fluorescent Activity Kit (Invitrogen™)

The P450 Demethylase Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of P450 demethylating actrivity in liver microsomes or cerosomes such as Cyp P450 3A4, 2B4 and 2D6.

This kit includes black 96-well plate(s), assay buffer, formaldehyde standard, and other components to perform the assay. This kit does not contain P450 systems. Microsome, cerosome, baculosome, or supersome P450 systems, or recombinant P450, need to be supplied by the user. A fluorescence 96-well microplate reader capable of reading fluorescent emission at 510 nm, with excitation at 450 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: demethylating P450 systems like liver microsomes or cerosomes such as Cyp P450 3A4, 2B4, and 2D6
• Standard curve range: 1.6 pmoles/100uL–20 pmoles/100uL
• Reactivity: human, rat

Background
The cytochromes P450 (P450s) are a superfamily of heme containing enzymes that display tremendous diversity with regard to substrate specificity and catalytic activity. P450s use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multicomponent electron transfer reactions. Catalysis by the eukaryotic P450 enzymes involves a multistep reaction cycle that includes two steps in which electron transfer is accomplished from a redox partner. The diflavin protein, NADPH cytochrome P450 reductase, contains both FAD and FMN and can transfer both electrons needed for the catalytic cycle3. In some P450 reactions, the second electron of the reaction cycle also can be delivered by cytochrome b5.

Lipid plays an important role in the reconstitution of P450-dependent activities after protein purification6. Most in vitro studies for the reconstitution of P450 activities use dilaurylphosphatidylcholine (DLPC) as the lipid component. The reconstitution of enzymatic activity involves a concentrated incubation of P450, its redox partners (NADPH and reductase), and lipid followed by dilution into the final assay components.

Assay principle
The P450 Demethylase Activity kit is designed to quantitatively measure the enzymatic activity of formaldehyde-producing enzymes such as cytochromes P450. The kit is unique in that the fluorescent substrate is not involved in the multicomponent P450 reaction, but measures the product of the demethylation, formaldehyde. No separation or washing is required. The kit has been validated for several P450 systems and should work with any biological system that is producing formaldehyde as a product of demethylation.

The kit provides an optimized buffer for P450, lyophilized vials of the cofactor NADPH for the reaction, a stable formaldehyde standard, the Formaldehyde Detection Reagent (FDR) and two 96-well plates for detecting the generated fluorescent signal. The end user will have to provide the microsomal, baculosome system or the recombinant P450, reductase and cytochrome b5 system and any cofactors necessary for activity, along with any candidate drugs, inhibitors, or activators being tested. The reaction should be carried out in our supplied buffer or a similar PBS based buffer system.

Following the P450 NADPH-induced reaction, the generation of formaldehyde can be stopped by addition of a suitable inhibitor, or the supplied stop solution of acetic acid. The FDR is then added to all the wells. If calibration to formaldehyde is needed (for cross lab comparisons) then a formaldehyde standard curve generated from the supplied standard should be run.

After a short incubation at 37°C for 30 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 450 nm. The P450 activity is determined based upon formaldehyde production.

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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

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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EnzChek™ Phospholipase A2 Assay Kit (Invitrogen™)

This kits takes our stand alone assay for phospholipase A2, PLA2 (A10072) and combines the necessary Ez and lipids to run 2 complete 96 well microplate assays that monitor activity in purified enzyme preparations and cell lysates. The EnzChek Phospholipase A2 Kit provides enough reagents for 2 microplates, using 200 µl volumes in 96 well format to perform continuous fluorometric monitoring of PLA2 . This product offers an alternative to our (B7701), (bis-BODIPY® FL C11-PC) reagent, by providing an PLA2 selective substrate and one that is ratiometric, thereby lowering variations produced by instrumentation and assay conditions. Phospholipase A2 or PLA2 represents a family of enzymes that hydrolyze the sn-2 ester linkage of phospholipids. The activities of these enzymes play important roles in cardiovascular, inflammatory and nervous system disorders, and in cancers. The EnzChek® Phospholipase A2 substrate provides sensitive and continuous rapid real-time monitoring of PLA2 enzyme activities. This unique substrate is selective for PLA2 and can be used either in a intensity or ratiometric based detection mode. In intensity based detection mode the PLA2 activity is monitored by the intensity increase of a single wavelength at approximately 515 nm. In ratiometric analysis, which is based on the distinct fluorescence resonance energy transfer (FRET) emission of this substrate prior to and after cleavage, PLA2 is detected by changes in the emission intensity ratio at 515/575 nm with excitation at ≈ 460 nm. Either detection mode provides a simple method with low background, high sensitivity and high specificity for PLA2.

Active Rap1 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Pierce Active Rap1 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Rap1 GTPase through specific protein interaction with the RalGDS protein-binding domain.

The Active Rap1 Pull-Down and Detection Kit includes purified GST-RalGDS Rap-binding domain (RBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Rap1 antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line that is known to have robust Rap1 activity.

Features of the Active Rap1 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Rap1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Rap1 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Rap1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
• Study active Rap1 signaling in cell junctions and adhesions
• Monitor Rap1 activity after stimulation with growth factors
• Screen small molecule inhibitors for their effects on Rap1 activity

The Active Rap1 Pull-Down and Detection Kit was validated for function and specificity of the active Rap1 enrichment method using cell lysates treated with GTPγS to activate endogenous Rap1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Rap1 in the GTP-bound form (active), resulting in a strong signal when endogenous Rap1 is present. GDP treatment pushes Rap1 into the GDP-bound state (inactive), resulting in minimal or no signal, regardless of Rap1 protein levels. This kit was 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.

Rap Background:
The Rap GTPases are part of the Ras family of GTPases and are encoded by Rap1a, Rap1b and Rap2. Rap GTPases are structurally similar to Ras GTPases and have similar effector and activator proteins, although Rap GTPases have different functional activities than Ras. While Ras is involved in cell proliferation and survival, Rap1 regulates cytoskeletal rearrangements, cell adhesion and cell junction formation.

The specificity of Rap1 and Ras is mediated by their respective upstream regulators and downstream effectors. The GEFs for Rap contain a CDC25 homology domain that mediates the GDP/GTP exchange reaction and a REM domain (Ras exchange motif). Some Rap GEFs include C3G, Epac1 and 2, RasGRP2, PDZ-GEF1 and 2 and PLCε. The binding domain used in this kit is RalGDS, a GEF that contains an RA domain to which Rap1 has a higher binding affinity than Ras. The RapGAPs, including Rap1GAP and the Spa-1 family, insert an asparagine side chain into the nucleotide-binding pocket to catalyze the GTP hydrolysis reaction. Rap effector proteins, including RAPL, Riam, AF-6, Krit1, RacGEFs, Tiam1, Vav2, Rho GAPs and ARAP3, are involved in cell-cell junctions and adhesion and are often localized to the membrane or at cell-cell junctions. Besides Ras, there is also cross-talk between Rap and Rho GTPases.

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

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

The Thermo Scientific Active Ras Pull-Down and Detection Kit enables selective enrichment and detection of GTP-bound Ras GTPase through a specific protein interaction with the Raf1 protein-binding domain.

The Active Ras Pull-Down and Detection Kit includes purified GST-Raf1 Ras-binding domain (RBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/washing buffer, anti-Ras antibody, SDS sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line known to have robust Ras activity.

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

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

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

The Active Ras Pull-Down and Detection Kit was validated for the function and specificity of the active Ras enrichment method using cell lysates treated with GTPγS to activate endogenous Ras and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment trap Ras in the GTP-bound, active form, resulting in a strong signal when endogenous Ras is present. GDP treatment pushes Ras into the GDP-bound, inactive state, resulting in little to no signal, regardless of Ras 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). Each 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 Ras using the GST-RBD protein and anti-Ras antibody supplied in the kit.

Ras Background:
The Ras superfamily of GTPases, named after the rat sarcoma viral oncogene, contains many Ras isoforms, including K-Ras, H-Ras, and N-Ras. While the three isoforms are expressed at different levels in different types of cells, in general, activating mutations of at least one of these isoforms are present in ~15% of all cancers. The Ras proteins serve as initiators of intracellular signal transduction from extracellular molecules and associate with the plasma membrane via lipid modification and prenylation of its carboxy terminus. These modifications at the carboxy terminus determine the localization of Ras to distinct membrane microdomains, which dictates subsequent downstream signaling. Ras signaling pathways affect many cellular processes including proliferation, survival, vesicular trafficking and gene expression.

Signaling through Ras is central to many cellular responses, and activation of Ras is regulated by several GEF and GAP proteins. The GEF proteins mediate GTPase activation by dissociating GDP from the inactive Ras to allow binding of Ras to GTP. Conversely, GAP proteins inactivate GTPases by hydrolyzing GTP to GDP. Ras mediates downstream signaling by interacting with effector proteins, including Raf and PI3 kinase. Raf1 is a serine/threonine protein kinase that is part of the MAP kinase kinase signaling pathway that leads to the activation of ERK and p38, which influences proliferation and survival. PI3 kinase signaling results in activation of AKT and mTOR, which are central for cell growth and survival. Ras is also integral for cellular differentiation and development, including immune cell development and function.

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

Glutathione Reductase Fluorescent Activity Kit (Invitrogen™)

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

This complete, ready-to-use kit includes black 96-well plate(s), glutathione reductase standard (200 mU/mL), glutathione reductase substrate, and other components to perform the assay. A 96-well microplate reader capable of reading the fluorescent emission at 510 nm, with excitation at 390 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: serum, plasma, RBCs, and cell lysates
• Sensitivity: 0.009 mU/mL
• Standard curve range: 0.15 mU/mL–5 mU/mL
• Reactivity: human

Background
Glutathione Reductase (GR) plays an indirect but essential role in the prevention of oxidative damage within the cell by helping to maintain appropriate levels of intracellular glutathione (GSH). GSH, in conjuction with the enzyme glutathione peroxidase (GP), is the acting reductant responsible for minimizing harmful hydrogen peroxide cellular levels. The regeneration of GSH is catalyzed by GR. GR is a ubiquitous 100-120 kDa dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using beta-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor.

Molecules such as NADPH act as hydride donors in a variety of enzymatic processes. NADPH has been suggested to also act as an indirectly operating antioxidant, given its role in the re-reduction of GSSG to GSH and thus maintaining the anti-oxidative power of glutathione. This assay has been validated for human serum, EDTA and heparin plasma, and isolated erythrocytes. Most cell lysates should also be compatible. GR activity varies across tissues and species, however this kit may measure GR activity from sources other than human.

Assay principle
The Glutathione Reductase Activity kit is designed to quantitatively measure Glutathione Reductase (GR) activity in a variety of samples. A GR 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 proprietary non-fluorescent detection reagent that will covalently bind to the free thiol group on GSH generated in the reduction of oxidized glutathione (GSSG) to yield a highly fluorescent product. After mixing the sample or standard with detection reagent and incubating at room temperature, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm

Related links
Learn more about ELISA kits
Learn more about other immunoassays

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.

Related Products
Pierce™ Kinase Enrichment Kit with ATP Probe
ActivX™ Desthiobiotin-ATP Probe
ActivX™ Desthiobiotin-ADP Probe