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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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Glutathione S-Transferase Fluorescent Activity Kit (Invitrogen™)

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

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

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

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

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

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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.

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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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.

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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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Amplex™ Red Catalase Assay Kit (Invitrogen™)

The Amplex Red Catalase Assay Kit provides a sensitive and simple fluorometric method for detecting as little as 50 mU/mL of catalase activity in a purified system in a 100µL assay volume.

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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EnzChek™ Lysozyme Assay Kit (Invitrogen™)

The EnzChek® Lysozyme Assay Kit provides researchers with a simple and sensitive assay to measure lysozyme activity in solution. The increase in fluorescence resulting from lysozyme activity is measured using a fluorometer or fluorescence microplate reader.

See our complete line of Fluorescence Microplate assays.

• Detect lysozyme activity concentrations as low as 20 U/mL
• Use standard fluorescein (FITC) excitation/emission settings
• Format allows for continuous measurement of lysozyme activity

The assay utilizes an innovative approach to measure lysozyme activity. Micrococcus lysodeikticus cell walls are used that have been extensively labeled with fluorescein causing quench of the fluorescence signal. Active lysozyme enzyme hydrolyzes the b-(1-4)-glucosidic linkages between the N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the mucopolysaccharide cell wall, relieving the quenching and yielding a dramatic increase in fluorescence that is proportional to lysozyme activity.

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.

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).

ActivX™ Azido-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 ActivX Azido-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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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™ 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.

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

Thermo Scientific Serine Hydrolase Probes are ActivX™ Fluorophosphonate (FP) and other tagged phosphonate probes to purify or assay serine hydrolase active sites using fluorescence, biotin-affinity, or mass spectrometry.

Features of the ActivX TAMRA-FP Serine Hydrolase Probe:

Specific—labels the reactive site of active serine hydrolases
Compatible—tags available for capture, detection and Staudinger conjugation
Flexible—use for in vitro or intracellular enzyme labeling

These ActivX™ FP Probes feature a reactive fluorophosphonate group that specifically and covalently labels the active-site serine of enzymatically active serine hydrolases. These probes are available with a desthiobiotin (biotin analog) tag for selective enrichment, TAMRA fluorophore for detection or a reactive- azido group (Staudinger reagents) that facilitates multiplex labeling when used with phosphine- or alkyne-derivatized tags. These probes can be used to assess activity or screen small molecule inhibitors against enzymes derived from cell lysates, subcellular fractions, tissues and recombinant proteins.

Applications:
• Determine serine hydrolase enzyme activity in cells and lysates
• Mapping the active-site serine of functionally diverse serine hydrolase family members (e.g. proteases, lipases, esterases)
• Screen for small molecule binding affinities and active-site inhibition
• Profile serine hydrolases using fluorescent, Western blot or mass spectrometry workflows

ActivX active-site probes are especially advantageous for determining active enzyme levels compared to other protein expression profiling techniques that only measure abundance. Because many of the proteolytic enzymes in the serine hydrolase family are expressed as inactive proenzymes (zymogens), the ActivX FP probes selective enrichment only those enzymes that are functionally active and biologically relevant at the time of labeling. This feature also makes it possible to perform selective screening of inhibitors or other conditions that alter enzyme activity.

Active serine hydrolases labeled with ActivX FP Probes can be detected and quantified by Western blot, fluorescent gel imaging or mass spectrometry by using a compatible tag. The TAMRA-FP probe can be used to label and detect active serine hydrolases in samples by fluorescent gel imaging, capillary electrophoresis or mass spectrometry. An anti-TAMRA antibody is also available for immuno-enrichment of TAMRA-FP probe-labeled proteins. The Azido-FP probe is used in combination with a phosphine- or alkyne-derivatized tag for either detection or enrichment. Desthiobiotin-FP probes can be used for streptavidin-based enrichment and detection of active serine hydrolase proteins in Western blotting or mass spectrometry.

The serine hydrolase superfamily is one of the largest, most diverse enzyme families in eukaryotic proteomes. Serine hydrolases are generally grouped into two large families: serine proteases (e.g., trypsin, elastase and thrombin) and metabolic serine hydrolases. Metabolic serine hydrolases are divided into multiple enzyme subclasses (e.g., esterases, lipases, amidases and peptidases) based on structure, catalytic mechanism and substrate preference.

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