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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.

Glutathione S-Transferase Fluorescent Activity Kit (Invitrogen™)

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

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

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

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

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

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

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.

LanthaScreen™ TR-FRET BACE1 Assay Kit

BACE1 (beta-secretase) is a key enzyme involved in the production of amyloid beta-peptides found in extracellular amyloid plaques of Alzheimer’s disease (AD). In some cases, early-onset familial AD can be attributed to a "Swedish"mutation in the amyloid precursor protein (APP), which dramatically enhances the cleavage of this protein by BACE1. This and other genetic and pathological evidence has led to therapeutic approaches that focus on the inhibition of BACE1 and other APP-cleaving enzymes, such as gamma-secretase.

Invitrogen’s LanthaScreen® TR-FRET BACE1 assay provides sensitive high-throughput screening for potential inhibitors of beta-secretase. The kit uses a terbium (Tb)-labeled anti-biotin antibody and a fluorescein-labeled BACE1-biotin substrate in a homogeneous TR-FRET assay format (Figure 1).



Contents and Storage:

The LanthaScreen® TR-FRET BACE1 Assay Kit contains BACE1 protein, fluorescently labeled BACE1 substrate, Tb-labeled anti-biotin antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or +4°C).

Amplex™ Acetylcholine/Acetlycholinesterase Assay Kit (Invitrogen™)

The Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit provides an ultrasensitive method for detecting acetylcholinesterase (AChE) activity in a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detect AChE activity levels as low as 0.002 U/mL in one hour
• Detect acetylcholine levels as low as 0.3 µM using excess AChE
• Achieve detection ranges of 0.3 µM to 100 µM acetylcholine
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

AChE activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. AChE converts the acetylcholine substrate to choline, which is then oxidized by choline oxidase to betaine and hydrogen peroxide. In the presence of horseradish peroxidase, hydrogen 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. Experiments with purified AChE from electric eel indicate that the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit can detect AChE levels as low as 0.002 U/mL using a reaction time of one hour. By providing an excess of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 µM, with a range of detection from 0.3 µM to 100 µM acetylcholine.

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™ Ultra Amylase Assay Kit (Invitrogen™)

The EnzChek® Ultra Amylase Assay Kit provides a solution-based assay featuring the speed, high sensitivity, and convenience required for measuring amylase activity or for screening amylase inhibitors in a high-throughput format. This EnzChek® kit contains a starch derivative—the DQ™ starch substrate—that is labeled with BODIPY® FL dye to such a degree that the fluorescence is quenched. This substrate is efficiently degraded by amylase; digestion relieves the quenching and yields highly fluorescent fragments. The accompanying increase in fluorescence is proportional to amylase activity and can be monitored with a fluorescence microplate reader or fluorometer, using standard fluorescein filters.

EnzChek® Ultra Amylase Assay Kit Specifications:
• Label (Ex/Em): BODIPY® FL conjugate (~502/512 nm)
• Kit contains lyophilized substrate, 10X reaction buffer, substrate solvent, a fluorescent standard, and a detailed protocol
• Sufficient reagents are supplied for 500 assays (using a 100 µL assay volume in a 96-well microplate assay format)


Find Fluorescent Substrates for Other Glycosidases
In addition to the EnzChek® Ultra Amylase Assay Kit, we offer kits and substrates to measure xylanase, lysozyme, β-galactosidase, and more. Review Detecting Glycosidases—Section 10.2 in the Molecular Probes® Handbook for more information on these products.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

EnzChek™ Elastase Assay Kit (Invitrogen™)

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

ActivX Desthiobiotin-GTP Probe (Thermo Scientific™)

Thermo Scientific Pierce ActivX Desthiobiotin-GTP Probe covalently label the active site of GTPases and GTPase subunits of G-protein coupled receptors enabling their selective enrichment thereby allowing identification and profiling of target enzyme classes across samples or to assess the specificity and affinity of enzyme inhibitors.

Features of ActivX Desthiobiotin-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 ActivX Desthiobiotin-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

ActivX Desthiobiotin-GTP Probe structure consists of a modified biotin attached to the nucleotide by a labile acyl-phosphate bond. After removal of GTP or GDP nucleotides from enzymes, the desthiobiotin-GTP probe can be used to covalently modify conserved lysine residues in the GTPase nucleotide-binding site. Desthiobiotin-GTP can selectively enrich, identify and profile target enzyme classes in samples. Pre-incubation of samples with small-molecule inhibitors that compete with active-site probes can be used to determine inhibitor binding affinity and target specificity.

Assessment of active-site labeling can be accomplished by either Western blot or mass spectrometry (MS). For the Western blot workflow, desthiobiotin-labeled proteins are enriched for SDS-PAGE analysis and subsequent detection with specific antibodies. For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested to peptides. Only the desthiobiotin-labeled, active-site peptides are enriched for analysis by LC-MS/MS. Both workflows can be used for determining inhibitor target binding, but only the MS workflow can identify global inhibitor targets and off-targets.

More Product Data
GTPase enrichment using a new active-site probe

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Pierce™ GTPase Enrichment Kit with GTP Probe

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
Learn more about ELISA kits
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™ Paraoxonase Assay Kit (Invitrogen™)

The EnzChek® Paraoxonase Assay Kit is a highly sensitive, homogenous fluorometric assay for the organophosphatase acitivty of paraoxonase. Based on the hydrolysis of a fluorogenic analog, this assay is >10-fold more sensitive than the colorimetric assay, and unlike the colorimetric assay, can distinguish samples of very similar paraoxonase activity. Under standard conditions, the assay requires only 5 µl of serum, yields a signal in as little as 15 minutes, and is linear for up to 50 minutes. The assay requires only a single reaction that can be either continuously monitored or terminated using the stop reagent included in the kit.

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.

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.

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

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.

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.

Related Products
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.

Related Products
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Acetylcholinesterase Fluorescent Activity Kit (Invitrogen™)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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KDalert™ GAPDH Assay Kit (Invitrogen™)

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

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

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

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

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

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

Butyrylcholinesterase Fluorescent Activity Kit (Invitrogen™)

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

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

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

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

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

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

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

The Active Cdc42 Pull-Down and Detection Kit includes purified GST-Pak1 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Cdc42 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line that is known to have robust Cdc42 activity.

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

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

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

The Active Cdc42 Pull-Down and Detection Kit was validated for the function and specificity of the active Cdc42 enrichment method using cell lysates treated with GTPγS to activate endogenous Cdc42 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Cdc42 in the GTP-bound, active form, resulting in a strong signal when endogenous Cdc42 is present. GDP treatment pushes Cdc42 into the GDP-bound, inactive state, resulting in minimal or no signal regardless of Cdc42 protein levels. This kit is optimized for Western blot detection with an HRP-conjugated secondary antibody (Goat Anti-mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

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

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

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P450 Demethylation Fluorescent Activity Kit (Invitrogen™)

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

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

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

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

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

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

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

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

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

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

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

Features of the ActivX Desthiobiotin-FP Serine Hydrolase Probe:

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

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

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

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

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

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

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

ActivX™ Desthiobiotin-ADP Probe (Thermo Scientific™)

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

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

EnzChek™ Phospholipase A2 Assay Kit (Invitrogen™)

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

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

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

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

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

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

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

The Active Rap1 Pull-Down and Detection Kit was validated for function and specificity of the active Rap1 enrichment method using cell lysates treated with GTPγS to activate endogenous Rap1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Rap1 in the GTP-bound form (active), resulting in a strong signal when endogenous Rap1 is present. GDP treatment pushes Rap1 into the GDP-bound state (inactive), resulting in minimal or no signal, regardless of Rap1 protein levels. This kit was optimized for Western blot detection using an HRP-conjugated secondary antibody (Goat Anti-rabbit IgG, Part No. 31460) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Glutathione Reductase Fluorescent Activity Kit (Invitrogen™)

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

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

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

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

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

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

Related links
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Learn more about other immunoassays

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

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

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

Features of the Pierce Kinase Enrichment Kit:

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

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

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

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

These products are subject to a limited use label license.

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

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.

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

ActivX™ Desthiobiotin-ATP Probe (Thermo Scientific™)

Individual ActivX Desthiobiotin-ATP Probe, 16 × 12.6µg
Molecular Weight: 1259.48
For use with the Pierce Kinase Enrichment kits.

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

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.