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Ethylene Glycol Thermo Scientific™

Thermo Scientific Pierce Ethylene Glycol Solution provides exceptional antifreeze protection and storage stability for antibody-enzyme conjugates because it is purified to remove impurities commonly found traditional glycerol stocks.

Features of Ethylene Glycol:

• Specially purified to remove impurities such as aldehydes, peroxides, iron and UV absorbing hydrocarbons
• Suitable for enzyme storage without the worry of losing enzymatic activity
• Stable for months

This product is a 50% (w/v) aqueous solution of highly purified ethylene glycol. When mixed in equal volume with purified protein samples, such as primary antibodies, the solution stabilizes and maintains the mixture as a liquid during freezer storage (-20°). Ethylene glycol is a suitable alternative to glycerol for most protein-storage applications. In fact, this preparation of ethylene glycol is devoid of various common glycerol impurities and potiential degradation products that are damaging to certain enzymes.

Novex™ ECL Chemiluminescent Substrate Reagent Kit Invitrogen™

Novex® ECL is a Chemiluminescent substrate used for chemiluminescence-based immunodetection of horse radish peroxidase (HRP) on western or dot blot membranes. It is a two-part reagent consisting of Reagent A (luminol) and Reagent B (an enhancer) used in equal volumes to attain the most intense light emission.

EZ-Link™ Maleimide Activated Horseradish Peroxidase Kit Thermo Scientific™

Thermo Scientific Pierce Maleimide Activated Horseradish Peroxidase Kit is for preparation of HRP conjugates with proteins, peptides or other ligands that contain sulfhydryl groups, such as reduced cysteines. It is sufficient fo the conjugation of 5 mg of immunoglobulin (IgG). The kit contains all buffers and one 10 mL desalting column.

Features of the Maleimide Activated Horseradish Peroxidase Kit :

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.

Related Products
EZ-Link™ Maleimide Activated Horseradish Peroxidase

Novex™ AP Mouse Chemiluminescent Detection Kit Invitrogen™

The Novex® AP Mouse Chemiluminescent Detection Kit provides superior chemiluminescent detection of proteins transferred to either nitrocellulose or PVDF membranes and treated with a protein-specific primary antibody. Detection is performed by treatment of the membrane with an alkaline phosphatase (AP)-conjugated anti-mouse secondary antibody followed by a ready-to-use chemiluminescent CDP-Star® substrate of alkaline phosphatase. Protein-specific signal is then captured by a chemiluminescent-compatible imaging system or X-ray film.

The Novex® AP Mouse Chemiluminescent Detection Kit is compatible with most western protocols but was designed for use with the iBind™ Western System.

Each kit includes a secondary AP-conjugated anti-mouse antibody, CDP-Star® chemiluminescent substrate, and an enhancer, sufficient for 10 mini-membranes processed using the iBind™ Western System.

Features include:

• Easy-to-use protocol
• High specificity, clean background
• Ultra-sensitivity
• Long-lasting signals—up to 5 days
• Results in less 5 minutes

EZ-Link™ Activated Peroxidase Antibody Labeling Kit Thermo Scientific™

The Thermo Scientific Pierce Activated Peroxidase and Antibody Labeling Kit is designed to prepare and purify HRP-conjugated antibodies with any previously unmodified primary or secondary antibody (IgG).

Features of the Activated Peroxidase and Antibody Labeling Kit:

Activated HRP – glutaraldehyde-activated horseradish peroxidase, ready for conjugation to antibodies and other proteins at sites of primary amines (e.g., lysines)
Permanent conjugation – reacts with primary amines to form covalent amide bonds; no reduction step is necessary to secure the linkage
High activity HRP – enzyme activity is greater than 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 in the kit is sufficient for reaction with 0.3 mg of IgG to produce about 0.5 mL of conjugate

Activated Peroxidase is HRP enzyme that has been activated with glutaraldehyde to contain reactive groups that will spontaneously conjugate with amines on an antibody (or other protein). Incubation of this Activated Peroxidase with IgG in sodium carbonate buffer (pH 9.5) results in formation of a conjugate where both the enzyme activity of HRP and the antigen-binding activity of IgG are preserved. The Antibody Labeling Kit includes the Activated Peroxidase and a Protein A/G column and buffers to purify the antibody conjugate from excess, unreacted HRP enzyme.

For additional information about glutaraldehyde conjugation, see our review of reductive amination and the discussion of applications that follows.

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.

Related Products
EZ-Link™ Plus Activated Peroxidase Kit

Novex™ AP Rabbit Chemiluminescent Detection Kit Invitrogen™

The Novex® AP Rabbit Chemiluminescent Detection Kit provides superior chemiluminescent detection of proteins transferred to either nitrocellulose or PVDF membranes and treated with a protein-specific primary antibody. Detection is performed by treatment of the membrane with an alkaline phosphatase (AP)-conjugated anti-rabbit secondary antibody followed by a ready-to-use chemiluminescent CDP-Star® substrate of alkaline phosphatase. Protein-specific signal is then captured by a chemiluminescent-compatible imaging system or X-ray film.

The Novex® AP Rabbit Chemiluminescent Detection Kit is compatible with most western protocols but was designed for use with the iBind™ Western System.

Each kit includes a secondary AP-conjugated anti-rabbit antibody, CDP-Star® chemiluminescent substrate, and an enhancer, sufficient for 10 mini-membranes processed using the iBind™ Western System.

Features include:

• Easy to use protocol
• High specificity, clean background
• Ultra-sensitivity
• Long-lasting signals--up to 5 days
• Results in less 5 minutes

Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit Invitrogen™

The Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit contains a sensitive, one-step assay that uses the Amplex® Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to detect hydrogen peroxide (H2O2) or peroxidase activity. The Amplex® Red reagent, in combination with horseradish peroxidase (HRP), has been used to detect H2O2 released from biological samples, including cells, or generated in enzyme-coupled reactions. Furthermore, Amplex® Red reagent can be used as an ultrasensitive assay for peroxidase activity when H2O2 is in excess. In the presence of peroxidase, the Amplex® Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin. Resorufin has excitation and emission maxima of approximately 571 nm and 585 nm, respectively, and because the extinction coefficient is high (58,000 ± 5,000 cm–1M–1), you can perform the assay fluorometrically or spectrophotometrically. This reaction has been used to detect as little as 10 picomoles of H2O2 in a 100 µL volume or 1 x 10–5 U/mL of HRP.

Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit Specifications:
• Ex/Em of reaction product resorufin: ~571/585 nm
• Sensitive one-step assay to detect hydrogen peroxide or peroxidase in vitro
• Can detect as little as 10 picomoles of hydrogen peroxide or 10 µU/mL of horseradish peroxidase activity in a 100 µL assay volume
• Provides sufficient reagents for approximately 500 assays using reaction volumes of 100 µL per assay
• Designed for a 96-well fluorescence or absorbance microplate reader, but can be easily adapted for other volumes, for fluorimeters, and for spectrophotometers


Find More Substrates for Oxidases
Review Substrates for Oxidases, Including Amplex Red Kits—Section 10.5 in the Molecular Probes® Handbook for more information on these products.

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

eBioscience™ Avidin HRP Invitrogen™

Avidin horseradish peroxidase is commonly used as a second step for the detection of biotinylated antibodies. Avidin is a glycoprotein from chicken egg whites that binds biotin with high affinity. Biotin is a tag that is frequently used to label antibodies and other probes for immunodetection methods.

This product is recommended for use in immunohistochemistry applications.

Conjugate
HRP

Reported Application
Immunohistochemical Staining

Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit Invitrogen™

The Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 488 nm laser of the flow cytometer. The Click-iT® Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

Multiplexable
The Click-iT® Plus formulation provides increased multiplexibility compared to the original Click-iT® EdU Flow Cytometry assays. Click-iT® Plus EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry, without loss of the accuracy or speed of the original Click-iT® EdU assay.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor® dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT® Plus EdU assay is compatible with cell cycle dyes. The Click-iT® Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

Quick and Simple Protocol
The Click-iT® Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

1. Treat cells with EdU.
2. Fix and permeabilize cells.
3. Detect S-phase cells with Click-iT® Plus detection cocktail for 30 min.
4. Wash once.
5. Analyze.

Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications.

Measure-IT™ Thiol Assay Kit Invitrogen™

The Measure-iT™ Thiol Assay Kit proves easy and accurate quantitation of thiol. The assay has a linear range of 0.05-5 uM thiol, making it up to 400 times more sensitive than colorimetric methods based on Ellman's reagent. The assay is performed at room temperature; maximum fluorescence signal is attained in 5 minutes and is stable for at least 1 hour.

Novex™ HRP Chromogenic Substrate (TMB) Invitrogen™

Novex® HRP Chromogenic Substrate is a ready to use single component reagent used for sensitive detection of a horse radish peroxidase (HRP) on western blot and dot blot membranes. Consisting of a ready-to-use solution of TMB, the Novex® HRP Chromogenic Substrate forms a blue precipatate up reaction with horse radish peroxidase (HRP).

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.

Related Products
EZ-Link™ Maleimide Activated Horseradish Peroxidase Kit

CellROX™ Deep Red Flow Cytometry Assay Kit Invitrogen™

The CellROX™ Deep Red Flow Cytometry Assay Kit enables the flow cytometric detection of reactive oxygen species (ROS) in live cells. The kit includes the novel, fluorogenic CellROX™ Deep Red Reagent, as well as SYTOX™ Blue dead cell stain, N-acetyl cysteine (an antioxidant, for negative control), and tert-butyl hydroperoxide solution (TBHP, an inducer of ROS).

View a selection guide for all CellROX® Reagents and Kits.

The CellROX™ Deep Red Flow Cytometry Assay Kit features:

• Fluorogenic probe formulated for flow cytometry that is oxidized in the presence of ROS
• Multicolor compatibility—minimal overlap with fluorophores excited by other laser lines, allowing easy multiplexing with other reagents
• Simple protocol—cells can be stained in complete media or other appropriate buffer, no need for serum free media

The CellROX™ Deep Red Detection Reagent is cell-permeable and non-fluorescent or very weakly fluorescent while in the reduced state. Upon oxidation, the reagent exhibits a strong fluorogenic signal that has absorption/emission maxima of 644/665 nm and remains localized in the cytoplasm. When used together with the included SYTOX™ Blue Dead Cell Stain, oxidatively stressed and non-stressed cells are reliably distinguished from dead cells by flow cytometry.

Pierce™ Conjugate Purification Kit Thermo Scientific™

The Thermo Scientific Pierce Conjugate Purification Kit is a gentle affinity chromatography system for purifying horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated antibodies from unreacted enzyme after labeling.

Features of the Conjugate Purification Kit:

Specific for IgG—procedure is designed for removal of HRP or AP enzymes from IgG antibodies; not for use with IgM, IgY, Fab fragments or streptavidin conjugates
Complete kit—includes affinity column, activator and elution buffers, desalting column and storage buffers for HRP and AP conjugates
High capacity—the single-use column-kit is designed for purification of 0.5 to 50 mg of antibody conjugate dissolved in Tris-buffered saline
Improves assays—removing unconjugated enzyme from antibody conjugates often reduces background and improves overal performance of immunoassays

This conjugate purification kit is designed to eliminate excess, unconjugated HRP or Alk-Phos enzyme from labeled antibodies (IgG) after performing antibody-enzyme conjugation reactions. The column of metal-chelate agarose resin binds to IgG-enzyme conjugates but allows non-conjugated enzyme (HRP or AP) to to pass through. A gentle, non-denaturing elution buffer recovers the fully active IgG-HRP or IgG-AP conjugate. The included desalting columns exchange the purified conjugate into any buffer of choice, ready for use in various immunodetection methods.

In this kit procedure, the nickel-chelated agarose binds IgG through a histidine-rich cluster on the Fc region at the junctures of the Cγ2 and Cγ3 domains, which is highly conserved across all mammalian IgGs. Purified IgG from sheep, mouse, goat, rat and rabbit will bind to nickel-chelated resin. Avidin or streptavidin enzyme conjugates cannot be purified by this method because they do not bind to the nickel-chelated agarose. Additionally, this kit cannot be used to purify antibodies from serum because other serum proteins will also bind to the nickel column.

Removing non-conjugated enzyme using this kit can improve assay results. Increase in assay sensitivity, shorter washes and incubation times, and a high signal-to-noise ratio can be more easily achieved when free enzyme is absent from the system. Furthermore, the column concentrates samples, which eases assay optimization when using conjugates in dilute solutions.
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