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EZ-Link™ Maleimide Activated Horseradish Peroxidase Thermo Scientific™

Thermo Scientific Pierce Maleimide Activated Horseradish Peroxidase is for preparation of HRP conjugates with proteins, peptides or other ligands that contain sulfhydryl groups, such as reduced cysteines. This product contains 5 mg of conjugated protein and can effectively modify 5 mg of immunoglobulin.

Features of Maleimide Activated Horseradish Peroxidase:

Activated HRP – horseradish peroxidase (HRP) modified with maleimide groups for conjugation to sulfhydryl molecules
Sulfhydryl-reactivemaleimide groups conjugate to reduced thiols (-SH), as in the side-chain of cysteine residues
High activity HRP – enzyme activity is greater than 240 units/mg; lyophilized, activated enzyme is stable for at least 12 months at 4°C
Complete kit – includes the activated HRP as well as two types of reagents for sulfhydryl-ready antibodies (IgG) or proteins for conjugation

This product consists of horseradish peroxidase (HRP) that has been modified with Sulfo-SMCC (Part No. 22322) to attach several maleimide groups per HRP molecule while retaining the peroxidase activity. The activated HRP will covalently attach to proteins or other molecule containing sulfhydryl groups (e.g., cysteines). HRP-conjugates of antibodies, proteins, peptides and other thiol-containing reporter probes are easily made using this method. The complete kit includes the activated HRP as well as two types of reagents for preparing sulfhydryl-ready antibodies (IgG) or proteins for conjugation.

The complete kit for Maleimide Activated Horseradish Peroxidase contains reagents for exposing or added the necessary sulfhydryl groups on antibodies (IgG) or practically any other protein. These general strategies are described briefly in the applications section of our review of Maleimide Reaction Chemistry. Of course, any protein that contains cysteines has sulfhydryl groups (-SH), but they must be reduced (not in the form of disulfide bonds) to be conjugated. Antibodies also contain disulfide bonds that can be targeted as antibody labeling sites; the hinge-region disulfide bonds in IgG can be selectively cleaved with the mild reducing agent 2-Mercaptoethylamine (Part No. 20408), which is included in the complete kit. Alternatively, sulfhydryl groups can be added to proteins (or any amine-containing molecule) using SATA reagent (Part No. 26102), which also is included in the kit.

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EZ-Link™ Maleimide Activated Horseradish Peroxidase Kit

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.

EZ-Link™ Plus Activated Peroxidase Thermo Scientific™

Thermo Scientific Pierce Plus Activated Peroxidase is an amine-reactive form of horseradish peroxidase (HRP) that provides coupling efficiencies of greater than 95% with antibodies and other proteins. This product consists of 5 mg of lyophilized HRP and is sufficient to modify 5 mg of Immunoglobulin (IgG)

Features of Thermo Scientific Pierce Plus Activated Peroxidase:

Activated HRP – periodate-treated, aldehyde-activated horseradish peroxidase, ready for conjugation to antibodies and other proteins at sites of primary amines (e.g., lysines)
Permanent conjugation – reacts efficiently (95%) with primary amines to form covalent amide bonds upon treatment with sodium cyanoborohydride (included in kit)
High activity HRP – enzyme activity is 120 to 200 units/mg; lyophilized, activated enzyme is stable for at least 12 months at -20°C
Convenient quantities – each 1 mg-quantity of activated enzyme is sufficient for reaction with 1 mg of IgG to produce about 0.5 mL of conjugate
Customizable – vary the molar ratios, reaction buffer and pH, and other parameters to acheive conjugates with different levels of HRP incorporation and activity

Plus Activated Peroxidase is horseradish peroxidase (HRP) enzyme, whose native carbohydrates (sugars) have been gently oxidized with periodate to produce amine-reactive aldehyde groups. These carbonyls of Plus Activated Peroxidase spontaneously and efficiently crosslink with primary amines on antibody or other proteins. The method is more effective than other amine-reactive chemistries, such as glutaraldehyde coupling, which often causes polymerization and a greater degree of conjugate inactivation. The pre-activated enzyme eliminates the difficulties inherent in preparing and validating activated peroxidase from scratch. The kit provides the enzyme, accessory reagents and protocol to easily produce high-performance conjugates for Western blotting, ELISA and other detection techniques.

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EZ-Link™ Plus Activated Peroxidase Kit

CellROX™ Orange Reagent, for oxidative stress detection Invitrogen™

CellROX® Orange Reagent is a novel fluorogenic probe for measuring oxidative stress in live cells. This cell-permeant dye is non-fluorescent while in a reduced state and exhibits bright orange fluorescence upon oxidation by reactive oxygen species (ROS), with absorption/emission maxima of ~ 545/565 nm. While this reagent is not formaldehyde-fixable, it can be multiplexed with other live cell-compatible probes such as GFP, CellEvent® Caspase 3/7 Green reagent, Image-iT® DEAD Green, Green and Deep Red MitoTracker® dyes, and NucBlue™ reagent.

CellROX® Orange Reagent is compatible with various platforms, such as traditional fluorescence microscopy, high content screening (HCS), and flow cytometery. This reagent is also compatible with the Tali™ Image-based Cytometer.

In addition, CellROX® Orange Reagent is:

• Compatible with other live cell probes, including GFP, and may be added directly to serum-containing growth media
• Provided as a stable, ready-to-use DMSO solution with a simple protocol compatible with standard workflows in fluorescence microscopy
• Provided as five single-use vials containing enough total reagent for five 96-well plates or 100 coverslips

Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge them. ROS play an important role in the progression of several diseases, including inflammation, atherosclerosis, aging, and age-related degenerative disorders.

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

Nitro blue Tetrazolium Chloride (NBT) Invitrogen™

Nitro blue tetrazolium chloride (NBT) can be used to assess cell viability as a function of redox potential. Actively respiring cells convert the water-soluble NBT to a deep blue-colored precipitate.

Measure-IT™ High-Sensitivity Nitrite Assay Kit Invitrogen™

Measure-iT assay kits bring add-and-read convenience and high-throughput scalability to the quantitation of nitrite, thiol, and lead/cadmium. The Measure-iT™ High-Sensitivity Nitrite Assay Kit features solid reproducibility over a wide quantitation range, as well as excellent tolerance of common sample contaminants. The assay is linear from 20 to 500 pmoles of nitrite, making it up to 50 times more sensitive than colorimetric assays based on the Griess reagent. The kit includes a nitrite quantitation standard and a protocol that's optimized for microplate-based application.

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.

CellROX™ Green Reagent, for oxidative stress detection Invitrogen™

CellROX® Green Reagent is a novel fluorogenic probe for measuring oxidative stress in live cells. The cell-permeant dye is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation by reactive oxygen species (ROS) and subsequent binding to DNA, with absorption/emission maxima of ~ 485/520 nm. This reagent can be formaldehyde-fixed and its signal survives detergent treatment, which makes it multiplexable with other compatible dyes and antibodies.

CellROX® Green Reagent is compatible with various platforms, such as traditional fluorescence microscopy, high content screening (HCS), flow cytometery, and microplate-based fluorimetry or high throughput screening (HTS). This reagent is also compatible with various benchtop instruments such as our FLoid™, Tali™, and Attune™ instruments.

In addition, CellROX™ Green Reagent is:

• Compatible with other live cell dyes, antibodies, and in cells expressing RFP
• Provided as a stable, ready-to-use DMSO solution with a simple no-wash protocol compatible with standard workflows in fluorescence microscopy
• Provided as five single-use vials containing sufficient total reagent for five 96-well plates or 100 coverslips

Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge them. ROS play an important role in the progression of several diseases, including inflammation, atherosclerosis, aging, and age-related degenerative disorders.

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

NBT/BCIP Reagent Kit Invitrogen™

The NBT/BCIP Reagent Kit includes 1 g samples of both NBT and BCIP for the chromogenic detection of phosphatase.

HPF (Hydroxyl Radical and Peroxynitrite Sensor) Invitrogen™

Aminophenyl fluorescein (APF, A36003) and hydroxyphenyl fluorescein (HPF), two reactive oxygen species (ROS) indicators developed by T. Nagano, offer greater specificity and stability than dichlorodihydrofluorescein diacetate (H2DCFDA, D399). APF and HPF show limited non-selective reactivity and relatively high resistance to light-induced oxidation. Both of these new fluorescein derivatives are nonfluorescent until they react with the hydroxyl radical or peroxynitrite anion; APF will also react with the hypochlorite anion. Together, these two ROS indicators can selectively detect hypochlorite anion. Upon oxidation, both APF and HPF exhibit bright green fluorescence (excitation/ emission maxima ~490/515 nm), making them compatible with any instrument that can detect fluorescein.

OxyBURST™ Green H2DCFDA, SE Invitrogen™

OxyBURST® Green H2DCFDA succinimidyl ester (2',7'-dichlorodihydrofluorescein diacetate, SE) is an amine-reactive assay reagent that can be used to prepare oxidation-sensitive conjugates of a wide variety of biomolecules and particles, including antibodies, antigens, peptides, proteins, dextrans, bacteria, yeast and polystyrene microspheres. Following conjugation to amines, the two acetates of OxyBURST® Green H2DCFDA can be removed by treatment with hydroxylamine at neutral pH to yield the dihydrofluorescein conjugate. OxyBURST® Green H2DCFDA conjugates are nonfluorescent until they are oxidized to the corresponding fluorescein derivatives.

ROS Indicator Specifications:
• Ex/Em: ~492–495/517–527 nm
• Product is air sensitive and should be stored under dry argon or nitrogen
• Product may be dissolved in DMSO, DMF, or ethanol for use
• Indicator is cell permeant (cell loading protocols are available in the literature)
• Fluorescence can be monitored using a flow cytometer, fluorometer, microplate reader, or fluorescence microscope, using excitation sources and filters appropriate for fluorescein


Find More ROS Indicators
We offer an assortment of Molecular Probes® products for the generation of reactive oxygen species (ROS), including singlet oxygen, superoxide, hydroxyl radical and various peroxide and hydroperoxides, as well as for their fluorometric detection in solution. Review Generating and Detecting Reactive Oxygen Species—Section 18.2 in the Molecular Probes® Handbook for more information on these products.

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

Dihydroethidium (Hydroethidine) Invitrogen™

The superoxide indicator dihydroethdium, also called hydroethidine, exhibits blue-fluorescence in the cytosol until oxidized, where it intercalates within the cell's DNA, staining its nucleus a bright fluorescent red.

EZ-Link™ Plus Activated Peroxidase Kit Thermo Scientific™

Thermo Scientific Pierce Plus Activated Peroxidase is an amine-reactive form of horseradish peroxidase (HRP) that provides coupling efficiencies of greater than 95% with antibodies and other proteins. Each kit contains 5 x 1 mg of lyophilized HRP and sufficient Sodium cyanoborohydride solution, quenching buffer, and buffers to conjugate 5 mg of Immunoglobulin (IgG)

Features of the Thermo Scientific Pierce Plus Activated Peroxidase Kit:

Activated HRP – periodate-treated, aldehyde-activated horseradish peroxidase, ready for conjugation to antibodies and other proteins at sites of primary amines (e.g., lysines)
Permanent conjugation – reacts efficiently (95%) with primary amines to form covalent amide bonds upon treatment with sodium cyanoborohydride (included in kit)
High activity HRP – enzyme activity is 120 to 200 units/mg; lyophilized, activated enzyme is stable for at least 12 months at -20°C
Convenient quantities – each 1 mg-quantity of activated enzyme is sufficient for reaction with 1 mg of IgG to produce about 0.5 mL of conjugate
Customizable – vary the molar ratios, reaction buffer and pH, and other parameters to acheive conjugates with different levels of HRP incorporation and activity

Plus Activated Peroxidase is horseradish peroxidase (HRP) enzyme, whose native carbohydrates (sugars) have been gently oxidized with periodate to produce amine-reactive aldehyde groups. These carbonyls of Plus Activated Peroxidase spontaneously and efficiently crosslink with primary amines on antibody or other proteins. The method is more effective than other amine-reactive chemistries, such as glutaraldehyde coupling, which often causes polymerization and a greater degree of conjugate inactivation. The pre-activated enzyme eliminates the difficulties inherent in preparing and validating activated peroxidase from scratch. The kit provides the enzyme, accessory reagents and protocol to easily produce high-performance conjugates for Western blotting, ELISA and other detection techniques.

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EZ-Link™ Plus Activated Peroxidase

Image-iT™ Lipid Peroxidation Kit, for live cell analysis Invitrogen™

The Image-iT® Lipid Peroxidation Kit enables the detection of lipid peroxidation in live cells through oxidation of BODIPY® 581/591 C11 reagent. This reagent localizes to membranes throughout live cells and upon oxidation by lipid hydroperoxides, displays a shift in peak fluorescence emission from ~590 nm to ~510 nm. Fluorescence from live cells shifts from red to green, providing a ratiometric indication of lipid peroxidation compatible with traditional and high content microscopy, as well as flow cytometry. This kit also includes cumene hydroperoxide as a positive control compound to induce lipid peroxidation in cells.

Additional Information:

• The reagent is provided as a stable, ready-to-use DMSO solution with a simple protocol compatible with standard workflows in fluorescence microscopy
• The reagent is provided as five single-use vials containing sufficient total reagent for five 96-well plates or 100 coverslips

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

Amplex™ Red Reagent Invitrogen™

The Amplex Red reagent, a highly sensitive and stable probe for H2O2, is our best fluorogenic substrate for peroxidase. In the presence of horseradish peroxidase (HRP), the Amplex Red reagent reacts in a 1:1 stoichiometry with H2O2 to produce highly fluorescent resorufin. Because H2O2 is produced in many different enzymatic reactions, the Amplex Red reagent can be used to detect the activity of many different enzymes.
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