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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

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 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.

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

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

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

BACE1 (β-Secretase) FRET Assay Kit, red

BACE1 (β-secretase) is a key enzyme involved in the production of Amyloid beta-peptides (Abeta) found in extracellular amyloid plaques of Alzheimers disease (AD). In some cases, early onset familial AD can be attributed to a "Swedish" mutation in the amyloid precursor protein (APP), dramatically enhancing the cleavage of this protein by BACE1. This and other genetic and pathological evidence has led to therapeutic approaches focusing on the inhibition of BACE1 and other APP-cleaving enzymes, such as gamma-secretase. The BACE1 fluorescence resonance energy transfer (FRET) Assay Kit provides a sensitive and efficient method for screening potential BACE1 inhibitors. This kit uses purified baculovirus-expressed BACE1 and a new red FRET peptide substrate based on the "Swedish" mutant.

The BACE1 FRET Assay Kit offers:

• Red emission to reduce compound interference
• Excitation at 545 nm and read emission at 585 nm
• An easy-to-use, rapid, homogeneous assay that is performed in solution
• Versatile formatting for high-throughput screening
• Stable results—measurements can be made for up to 24 hours

The principle of the BACE1 FRET assay is as follows: The peptide substrate is synthesized with two fluorophores, a fluorescent donor (a rhodamine (Rh) derivative), and a proprietary quenching acceptor. The distance between these two groups has been selected so that upon light excitation, the donor (D) fluorescence energy is significantly quenched by the acceptor (A) through a quantum mechanical phenomenon known as resonance energy transfer (Figure 1). Upon cleavage by the protease, the fluorophore is separated from the quenching group, restoring the full fluorescence yield of the donor. Thus, a weakly fluorescent peptide substrate becomes highly fluorescent upon enzymatic cleavage; the increase in fluorescence is linearly related to the rate of proteolysis.

Source of BACE1 Enzyme:
A cDNA sequence encoding amino acids 1-460 of human BACE1, corresponding to the ectodomain, was expressed in recombinant, baculovirus-infected insect cells. Purified BACE1 exists as the proBACE1 form having an apparent molecular weight of 55 kDa and an N-terminal sequence of TQHGIRLPLR.

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.

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

EnzChek™ Ultra Xylanase Assay Kit (Invitrogen™)

The EnzChek® Ultra Xylanase Assay Kit features a quick and convenient mix-and-read format for the detection and monitoring of xylanase activity. The increase in fluorescence resulting from xylanase activity is measured using a fluorometer or fluorescence microplate reader.

See our complete line of Fluorescence Microplate assays.

• Detect concentrations of xylanase activity as low as 1.5 mU/mL
• Suitable for a broad pH range (pH 4 to 10)
• Format allows for continuous detection of xylanase activity
• Excitation/emission maxima of ~358/455 nm, well suited for DAPI filter settings

The EnzChek® Ultra Xylanase Assay Kit provides the speed, sensitivity, and convenience required for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format. The hydrolysis of xylosidic linkages within the included Xylanase Substrate (hemicellulose polysaccharides) results in the unquenching of the attached fluorescent dyes. This kit can be used for continuous detection of xylanase activity, and offers broad dynamic and pH ranges. Each kit contains sufficient substrate for ~500 assays in a 96-well microplate format. Additionally, the kit contains a fluorescent reference standard that can be used to quantify the xylanase activity.

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).

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

Related Products
ActivX Desthiobiotin-GTP Probe

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
Learn more about ELISA kits
Learn more about other immunoassays

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.

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.

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.

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.

Ceruloplasmin Colorimetric Activity Kit (Invitrogen™)

The Ceruloplasmin Activity research-use-only kit is a colorimetric activity assay designed for the quantification and detection of ceruloplasmin activity in serum and urine samples.

This complete, ready-to-use kit includes clear 96 well plate(s), ceruloplasmin standard (200 Units/mL), ceruloplasmin substrate, and other components to perform the assay. A 96-well microplate reader capable of reading optical density at 560 nm is required for use of this kit.

Performance characteristics
• Assay type: colorimetric activity kit
• Sample types: serum and urine samples
• Sensitivity: 3.26 mU/mL
• Standard curve range: 31.2 mU/mL–1,000 mU/mL
• Reactivity: species independent

Background
Ceruloplasmin (Cp) is a multicopper oxidase enzyme involved in the safe handling of oxygen in some metabolic pathways of vertebrates. Discovered in 1948, a blue protein from the a2-globulin fraction of human serum possessing oxidase activity towards aromatic diamines and catechol was purified by Holmberg and Laurell. It was denoted ceruloplasmin, literally meaning "a blue substance from plasma". Specialized copper sites have been recruited during evolution to provide long-range electron transfer reactivity and oxygen binding and activation in proteins destined to cope with oxygen reactivity in different organisms.

Ceruloplasmin belongs to the family of multicopper oxidases which are among the few enzymes able to bind molecular oxygen to perform its complete reduction to water. Ceruloplasmin contains 95% of the copper in serum. Cp found in serum is expressed in the liver, but it is also expressed in the brain, lung, spleen, and testis. This assay has been validated for serum and urine samples. Ceruloplasmins are ancient enzymes that should behave in a similar manner to the colorimetric substrate. This assay should to measure Cp activity from a wide range of sources.

Assay principle
The Ceruloplasmin Activity kit is designed to quantitatively measure Ceruloplasmin activity in diluted serum and urine samples. A human ceruloplasmin 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. The reconstituted ceruloplasmin substrate is added and the plate is incubated at 30°C for 60 minutes. The ceruloplasmin in the standards and samples reacts with the substrate to produce a colored product. The optical density is read at 560 nm. Increasing levels of ceruloplasmin in the samples causes an increase in the fuchsia (pink-purple) product.

Ceruloplasmin unit definition
Ceruloplasmin activity is based upon the published determination by G. Curzon and L. Vallet, Biochem. J., 1960, 74:279-287. Due to our optimized reagents and incubation times the optical density generated by 1 U/mL of ceruloplasmin is substantially higher than 0.1 OD.

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