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NA-XTD™ Influenza Neuraminidase Assay Reagent Set, no plates included Invitrogen™

The NA-XTD™ Influenza Neuraminidase Assay Kit is a next-generation chemiluminescence-based assay that provides a longer signal read-out compared to the first-generation NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit. The NA-XTD™ kit includes detection reagents and microplates, eliminating the need for luminometers equipped with a reagent injector, and improving ease-of-use. This kit also includes complete assay protocols for quantitating sensitivity to neuraminidase inhibitors in various influenza virus isolates including: human influenza types A and B, A⁄H1N1 pandemic, avian, equine and porcine viruses.

Key product features:
• Superior performance—improved, long-lived light emission kinetics and read-time flexibility
• High sensitivity—high assay signal-to-noise, detection of low-virus concentrations, and wide assay dynamic range
• Simple instrumentation requirement—no reagent injectors or luminometer needed
• Multiple applications—neuraminidase inhibitor IC50 assays and cell-based virus quantitation

Long-Lived Light Emission Kinetics & Read-Time Flexibility
The NA-XTD™ assay yields much longer-lived light emission kinetics than does the NA-Star® assay, eliminating the need for luminometer instrumentation with reagent injectors and enabling read-time flexibility and batch-mode processing of assay plates. NA-XTD™ assay signal half-life is approximately 2 hours, longer than the 5-10 minute half-life of the NA-Star® assay signal. IC-50 values determined from data collected immediately or up to 3 hours following addition of NA-XTD™ Accelerator solution are identical (Figure 1).

High Sensitivity
The NA-XTD™ chemiluminescent assay provides higher detection sensitivity (better low-end detection limit), higher assay signal-to-noise ratio, and wider assay dynamic range than fluorescent assays with the MUNANA substrate. The NA-XTD™ assay also typically demonstrates slightly higher signal-to-noise than the NA-Star® assay. The NA-XTD™ assay provides 2–50-fold higher sensitivity by signal-to-noise ratio than MUNANA-based fluorescence assays, depending on the virus isolate (Figure 2). The NA-XTD™ assay provides a dynamic range of detection of 3–4 orders of magnitude of neuraminidase enzyme concentration, compared to 2–3 orders of magnitude range with fluorescent MUNANA assays. The wide chemiluminescent neuraminidase assay range enables determination of IC-50 values over a wide range of virus concentrations, eliminating the need to titer virus prior to performing IC50 determination assays (Figure 3).

Simple Instrumentation Requirement
The NA-XTD ™ assay can be used for virus quantitation in media samples from 96-well microplates or other virus cultures for monitoring viral growth or infection, or for performing viral inhibition assays in a cell-based system. Optimally, a small sample of culture media is removed and assayed directly with NA-XTD™ reagents, permitting multiple samples to be assayed over time (Figure 4).

Multiple Applications in One Complete Kit
The NA-XTD™ Assay Buffer is used as diluent for virus samples, neuraminidase inhibitors and NA-XTD™ substrate. An optional NA Sample Prep Buffer is also included for Triton® X-100 detergent addition to virus preparations, which increases NA activity in some virus preparations. The next-generation NA-XTD™ Accelerator solution triggers high intensity light emission from the NA-XTD™ reaction product. For added convenience and assay performance, the kit includes NA-Star™ Detection Microplates. These 96-well solid white assay microplates were selected for optimum assay performance, including high signal intensity, low background, and minimum well-to-well cross-talk. The kit includes a comprehensive assay protocol that provides virus and neuraminidase inhibitor (NI) dilution recommendations, a recommended plate layout for NI sensitivity assays, a protocol for virus quantitation, and a literature reference list. The NA-XTD™ Influenza Neuraminidase Assay Kit is compatible with a wide range of luminometers, including single-mode and multi-mode instruments, with no need for injectors.

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

NA-XTD™ Influenza Neuraminidase Assay Kit Invitrogen™

The NA-XTD™ Influenza Neuraminidase Assay Kit is a next-generation chemiluminescence-based assay that provides a longer signal read-out compared to the first-generation NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit. The NA-XTD™ kit includes detection reagents and microplates, eliminating the need for luminometers equipped with a reagent injector, and improving ease-of-use. This kit also includes complete assay protocols for quantitating sensitivity to neuraminidase inhibitors in various influenza virus isolates including: human influenza types A and B, A⁄H1N1 pandemic, avian, equine and porcine viruses.

Key product features:
• Superior performance—improved, long-lived light emission kinetics and read-time flexibility
• High sensitivity—high assay signal-to-noise, detection of low-virus concentrations, and wide assay dynamic range
• Simple instrumentation requirement—no reagent injectors or luminometer needed
• Multiple applications—neuraminidase inhibitor IC50 assays and cell-based virus quantitation

Long-Lived Light Emission Kinetics & Read-Time Flexibility
The NA-XTD™ assay yields much longer-lived light emission kinetics than does the NA-Star® assay, eliminating the need for luminometer instrumentation with reagent injectors and enabling read-time flexibility and batch-mode processing of assay plates. NA-XTD™ assay signal half-life is approximately 2 hours, longer than the 5-10 minute half-life of the NA-Star® assay signal. IC-50 values determined from data collected immediately or up to 3 hours following addition of NA-XTD™ Accelerator solution are identical (Figure 1).

High Sensitivity
The NA-XTD™ chemiluminescent assay provides higher detection sensitivity (better low-end detection limit), higher assay signal-to-noise ratio, and wider assay dynamic range than fluorescent assays with the MUNANA substrate. The NA-XTD™ assay also typically demonstrates slightly higher signal-to-noise than the NA-Star® assay. The NA-XTD™ assay provides 2–50-fold higher sensitivity by signal-to-noise ratio than MUNANA-based fluorescence assays, depending on the virus isolate (Figure 2). The NA-XTD™ assay provides a dynamic range of detection of 3–4 orders of magnitude of neuraminidase enzyme concentration, compared to 2–3 orders of magnitude range with fluorescent MUNANA assays. The wide chemiluminescent neuraminidase assay range enables determination of IC-50 values over a wide range of virus concentrations, eliminating the need to titer virus prior to performing IC50 determination assays (Figure 3).

Simple Instrumentation Requirement
The NA-XTD ™ assay can be used for virus quantitation in media samples from 96-well microplates or other virus cultures for monitoring viral growth or infection, or for performing viral inhibition assays in a cell-based system. Optimally, a small sample of culture media is removed and assayed directly with NA-XTD™ reagents, permitting multiple samples to be assayed over time (Figure 4).

Multiple Applications in One Complete Kit
The NA-XTD™ Assay Buffer is used as diluent for virus samples, neuraminidase inhibitors and NA-XTD™ substrate. An optional NA Sample Prep Buffer is also included for Triton® X-100 detergent addition to virus preparations, which increases NA activity in some virus preparations. The next-generation NA-XTD™ Accelerator solution triggers high intensity light emission from the NA-XTD™ reaction product. For added convenience and assay performance, the kit includes NA-Star™ Detection Microplates. These 96-well solid white assay microplates were selected for optimum assay performance, including high signal intensity, low background, and minimum well-to-well cross-talk. The kit includes a comprehensive assay protocol that provides virus and neuraminidase inhibitor (NI) dilution recommendations, a recommended plate layout for NI sensitivity assays, a protocol for virus quantitation, and a literature reference list. The NA-XTD™ Influenza Neuraminidase Assay Kit is compatible with a wide range of luminometers, including single-mode and multi-mode instruments, with no need for injectors.

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

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

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.

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.

Superoxide Dismutase (SOD) Colorimetric Activity Kit Invitrogen™

The Superoxide Dismutase Activity research-use-only kit is a colorimetric activity assay designed for the quantification and detection of superoxide dismutase activity in serum, plasma, cells, tissues and erythrocyte lysates.

This complete, ready-to-use kit includes clear 96-well plate(s), superoxide dismutase standard (1 Unit/vial), superoxide dismutase substrate, and other components to perform the assay. A 96-well microplate reader capable of reading optical density at 450 nm is required for use of this kit.

Performance characteristics
• Assay type: colorimetric activity kit
• Sample types: serum, plasma, cells, tissues, and erythrocyte lysates
• Sensitivity: 0.044 U/mL
• Standard curve range: 0.06 U/mL–4 U/mL
• Reactivity: species independent

Background
Short-lived and highly reactive oxygen species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide are continuously generated in vivo. In the resting state, the balance between antioxidants and oxidants is sufficient to prevent the disruption of normal physiologic functions; however, either increases in oxidants or decreases in antioxidants can disrupt this balance giving rise to elevated levels of reactive oxygen species (ROS). The cellular levels of ROS are controlled by antioxidant enzymes and small molecule antioxidants. The major antioxidant enzymes, superoxide dismutases (SODs), including copper-zinc superoxide dismutase (Cu/ZnSOD, SOD1), manganese superoxide dismutase (MnSOD, SOD2) and extracellular superoxide dismutase (EC-SOD, SOD3), all play critical roles in scavenging superoxide .

Decreased SOD activity results in elevated level of superoxide which in turn leads to decreased NO but increased peroxynitrite concentrations. The major intracellular SOD is a 32-kD copper and zinc containing homodimer (Cu/Zn SOD). The mitochondrial SOD (MnSOD) is a manganese-containing 93-kD homotetramer that is synthesized in the cytoplasm and translocated to the inner matrix of mitochondria. This assay has been validated for serum and urine samples. Superoxide dismutases are ancient enzymes that should behave in a similar manner to the colorimetric substrate. This assay should to measure SOD activity from a wide range of sources.

Assay principle
The Superoxide Dismutase Activity kit is designed to quantitatively measure SOD activity in a variety of samples. The assay measures all types of SOD activity, including Cu/Zn, Mn, and FeSOD types. A bovine erythrocyte SOD standard is provided to generate a standard curve for the assay and all samples should be read off of the standard curve. Samples are diluted in our specially colored sample diluent and added to the wells. The substrate is added followed by Xanthine Oxidase Reagent and incubated at room temperature for 20 minutes. The xanthine oxidase generates superoxide in the presence of oxygen, which converts a colorless substrate in the detection reagent into a yellow colored product. The colored product is read at 450 nm. Increasing levels of SOD in the samples causes a decrease in superoxide concentration and a reduction in yellow product.

SOD unit definition
One unit of SOD is defined as the amount of enzyme causing half the maximum inhibition of the reduction of 1.5 mM Nitro blue tetrazolium in the presence of riboflavin at 25°C and pH 7.8.

Related links
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Learn more about other immunoassays

EnzChek™ Gelatinase/Collagenase Assay Kit, 250-2,000 assays Invitrogen™

The EnzChek Gelatinase/Collagenase Assay Kit provides a sensitive, convenient and fast fluorometric method for measuring gelatinase or collagenase 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 fluorescein-labeled DQ gelatin conjugate that is highly labeled so that the fluorescence signal is quenched until enzymatic digestion yields highly fluorescent fragments. This substrate is also available separate from the kit (D-12054).

KDalert™ GAPDH Assay Kit with Manual 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.

Catalase Colorimetric Activity Kit Invitrogen™

The Catalase Activity research-use-only kit is a colorimetric activity assay designed for the quantification and detection of catalase activity in serum, plasma, cells, tissues and erythrocyte lysates.

This complete, ready-to-use kit includes clear 96 well plate(s), catalase standard (100 Unit/mL), catalase substrate, and other components to perform the assay. A 96-well microplate reader capable of reading optical density at 560 nm (acceptable range: 540-580 nm) is required for use of this kit.

Performance characteristics
• Assay type: colorimetric activity kit
• Sample types: serum, plasma, cells, tissues, and erythrocyte lysates.
• Sensitivity: 0.052 U/mL
• Standard curve range: 0.15 U/mL–5.0 U/mL
• Reactivity: species independent

Background
Hydrogen peroxide is one of the most frequently occurring reactive oxygen species. It is formed either in the environment or as a by-product of aerobic metabolism, superoxide formation and dismutation, or as a product of oxidase activity. Both excessive hydrogen peroxide and its decomposition product hydroxyl radical, formed in a Fenton-type reaction, are harmful for most cell components. Its rapid removal is essential for all aerobically living prokaryotic and eukaryotic cells. However, hydrogen peroxide can act as a second messenger in signal transduction pathways, in immune cell activation, inflammation processes, cell proliferation, and apoptosis.

One of the most efficient ways of removing peroxide is through the enzyme catalase, which is encoded by a single gene and is highly conserved among species. Mammals, including humans and mice, express catalase in all tissues, and a high concentration of catalase can be found in the liver, kidneys, and erythrocytes. The expression is regulated at transcription, post-transcription, and post-translation levels. High catalase activity is detected in peroxisomes. More recently, short wavelength UV radiation has been shown to produce reactive oxygen species (ROS) through the action of catalase. This response is thought to act as a mechanism to protect DNA by converting damaging UV radiation into ROS species that can be metabolized and detoxified by cellular antioxidant enzymes. This assay has been validated for serum, EDTA and heparin plasmas from a variety of species.

Assay principle
The Catalase Activity kit is designed to quantitatively measure catalase activity in a variety of samples. A bovine catalase standard is provided to generate a standard curve for the assay and all samples should be read off of the standard curve. Samples are diluted in the provided Assay Buffer and added to the wells of a half area clear plate. Hydrogen peroxide is added to each well and the plate incubated at room temperature for 30 minutes. The supplied Colorimetric Detection Reagent is added, followed by diluted horseradish peroxidase, and incubated at room temperature for 15 minutes. The HRP reacts with the substrate in the presence of hydrogen peroxide to convert the colorless substrate into a pink-colored product. The colored product is read at 560 nm. Increasing levels of catalase in the samples causes a decrease in hydrogen peroxide concentration and a reduction in pink product.

Catalase unit definition
One unit of Catalase decomposes one micromole of hydrogen peroxide per minute at 25°C and pH 7.0.

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Learn more about other immunoassays

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.

EnzChek™ Reverse Transcriptase Assay Kit Invitrogen™

The EnzChek® Reverse Transcriptase Assay Kit is a convenient, efficient, and inexpensive assay for measuring reverse transcriptase activity. In less than an hour, samples can be read in a fluorometer or microplate reader with filter sets appropriate for fluorescein (FITC).

See our complete line of Fluorescence Microplate assays.

• Detect as little as 0.02 units of HIV-1 reverse transcriptase
• Large dynamic assay range, detect up to a 50-fold linear range
• Simple to use assay, amenable for automated high-throughput screening applications

The EnzChek® Reverse Transcriptase Assay Kit uses PicoGreen® reagent, which preferentially detects dsDNA or RNA-DNA heteroduplexes over single-stranded nucleic acids or free nucleotides. In this assay, the reverse transcriptase activity in a biological sample generates long RNA-DNA heteroduplexes from a mixture of a long poly(A) template, an oligo-dT primer, and dTTP. The RNA-DNA heteroduplexes formed are then detected by the PicoGreen® reagent.

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

EnzChek™ Direct Phospholipase C Assay Kit Invitrogen™

This kit provides continuous monitoring of phosphotidyl choline specific phospholipase C (PLC) activity in microplate based biochemical assays. The assay uses a PCL selective fluorogenic substrate that emits a bright green emission upon cleavage.

The EnzChek® Direct Phospholipase C Assay Kit provides a simple and robust method for monitoring PC-PLC activity. Each kit provides enough reagents for 2 microplates, using 200 µl volumes in 96 well format. PC-PLC plays a crucial role in many cell signaling pathways involved in apoptosis and cell survival, as well as diseases ranging from cancer to HIV1-7. The assay uses a glycerophospho-ethanolamine with a dye-labeled sn-2 acyl chain as a substrate for PC-PLC. Substrate cleavage by PC-PLC before the phosphate releases the dye-labeled diacylglycerol, which produces a positive fluorescence signal that may be measured continuously. The reaction product has absorption and fluorescence emission maxima of 509 nm and 516 nm, respectively. Using purified enzyme from Bacillus cereus, the assay can detect as little as 10 mU⁄mL PC-PLC after one hour at room temperature. The kit has been proven useful for characterizing PC-PLC inhibition, and since it offers a direct measurement, the potential for false positives in a compound screen is eliminated.

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.
.
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.

Related links
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Learn more about other immunoassays

Histone Demethylase Fluorescent Activity Kit Invitrogen™

The Histone Demethylase Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of histone demethylase activity of lysine-specific histone demethylase 1 (LSD1)-and Jumonji-type demethylases.

This kit includes black 96-well plate(s), LSD1/JMJD2A assay buffers, formaldehyde standard, and other components to perform the assay. This kit does not contain demethylase enzyme samples. A source of LSD1-type or Jumonji-type demethylase, along with any cofactors, enzyme substrates, inhibitors, and/or activators 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: lysine-specific histone demethylase 1 (LSD1) and Jumonji-type demethylases
• Standard curve range: 0.128 uM–0.64 uM for LSD1, 2.5 uM-10uM for JMJD2A
• Reactivity: human

Background
Histone Demethylase (HDM) catalyzes the site-specific demethylation of methyl-lysine residues in histones to dynamically regulate chromatin structure, gene expression, and potentially other genomic functions. Lysine-specific HDMs were first discovered in 2004 and are currently among the most actively studied formaldehyde-producing enzymes. At present, there are two known classes of HDMs: the flavin adenine dinucleotide (FAD)-dependent Lysine Specific Demethylase 1 (LSD1) family and the Fe(II)-dependent Jumonji C (JmjC) family. Although the LSD1 and JmjC HDMs employ different cofactors and catalytic mechanisms, both produce formaldehyde as a byproduct of the demethylation reaction.

Despite their biological importance, HDMs have proven difficult to quantitatively assay owing to their relatively low turnover numbers, hindering understanding of their kinetic properties, substrate specificities, and reaction mechanisms. This assay has been validated for lysine-specific histone demethylase 1 (LSD1)-and Jumonji-type demethylases. For HDM samples in cell lysates, we include a specially formulated Cell Lysis Buffer, that has been shown not to interfere with formaldehyde detection. Cell lysis buffers containing SDS and Triton X-100 inhibit the formaldehyde signal reaction and should not be used.

Assay principle
The Histone Demethylase Activity kit is to quantitatively measure the enzymatic activity of formaldehyde-producing enzymes such as histone demethylases. The kit is unique in that the product of these enzymatic demethylation reactions, formaldehyde, is quantitated directly by a fluorescent product. No separation or washing is required. The kit has been validated for both LSD1 and JMJD2A histone demethylases (HDMs).

The kit provides optimized buffers for the HDMs LSD1 and JMJD2A, a stable formaldehyde standard, the Formaldehyde Detection Reagent (FDR), and two 96-well plates for detecting the generated fluorescent signal. The kit allows any enzymatic reaction generating formaldehyde to be measured. The end user will have to provide the demethylase system and any cofactors necessary for activity, along with any test inhibitors or activators. The kit allows end users to produce HDM activity in many in vivo and in vitro systems and then determine the activity by measuring formaldehyde generation. For in vitro studies, the HDM reaction should be carried out in our supplied buffers using optimized reaction conditions for the demethylation.

Following the formaldehyde generating reaction, the reaction can be stopped by addition of a suitable inhibitor. 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.

Related links
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