HRP, a well-known and commercially available oxidoreductase, is a 44 kDa glycoprotein with six lysine residues, which is extensively used. It is a highly valuable tool in research and has been employed in a wide range of applications.
HRP can be conjugated to antibodies and proteins. Due to its high turnover rate, secondary antibodies conjugated to this enzyme can rapidly produce robust signals. Moreover, HRP substrates can be categorized as chromogenic (eg, DAB, TMB, OPD), fluorogenic (eg, ADHP), or chemiluminescent (eg, ECL), depending on whether they yield a colored, fluorescence signal, or a luminescent signal, respectively. Our recommended HRP-conjugated secondary antibodies include goat anti-mouse IgG, goat anti-rabbit IgG, rabbit anti-human IgG, goat anti-donkey IgG, goat anti-rat IgG, and goat anti-chicken IgY. These antibodies are carefully selected to ensure high specificity and reliable detection in immunoassays.
Our HRP-conjugated antibodies are widely used in western blotting (WB), immunoprecipitation (IP), dot blot (Dot), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry on frozen (IHC-Fr) and paraffin-embedded (IHC-P) tissues, ensuring reliable and sensitive detection across various applications. Additionally, our HRP Conjugation Kit is designed to enable quick and straightforward labeling of primary antibodies with HRP for use in a variety of immunoassays. The kit is provided in several formats to accommodate different antibody amounts, from 30 µg to 1 mg, ensuring flexibility for both small-scale and large-scale experiments. For optimal results, the antibody should be purified, and buffer components that can interfere with the conjugation reaction (such as amino acids or thiols) should be removed beforehand.
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Features
- High sensitivity: Optimized for detecting low-abundance targets, delivering high signal-to-noise ratios.
- Signal amplification: HRP-conjugated secondary antibodies amplify detection signals by catalyzing substrate oxidation, generating either a visible color change with chromogenic substrates or light emission with chemiluminescent substrates.
- Robust performance: Effective in dilutions ranging from 1:2,000 to 1:20,000 and providing high specificity with minimal background.
- Broad substrate compatibility: Supports chromogenic, fluorogenic, and chemiluminescent detection.
- Extensive validation: Thoroughly tested and optimized for various applications.
Applications
- Western blot: Enables protein detection and characterization in complex mixtures after gel electrophoresis and transfer, with HRP used for signal visualization with several chromogens.
- Dot blot: Enables rapid detection and semi-quantitative analysis of target proteins by spotting samples on a membrane, followed by HRP-mediated signal development.
- Enzyme-linked immunosorbent assay (ELISA): Allows for sensitive and specific measurement of proteins or other antigens in samples through colorimetric or chemiluminescent signals generated by the enzyme-conjugated antibody.
- Immunocytochemistry: Allows localization and visualization of proteins in cultured cells, leveraging the colorimetric or chemiluminescent signal produced by HRP.
- Immunohistochemistry: Used for detecting antigens in fresh, paraffin-embedded, and frozen tissues.
- Immunoprecipitation: Following isolation and concentration of the specific target proteins from complex samples, detection of your target protein can be performed using HRP-conjugated antibodies.
What is the HRP conjugate?
An HRP conjugate is an antibody or other molecule linked to a horseradish peroxidase protein, an enzyme that catalyzes substrate reactions to produce a detectable signal in immunoassays.
How do HRP-conjugated antibodies perform in high-throughput screening applications?
HRP-conjugated antibodies excel in high-throughput screening by delivering robust, sensitive, and rapid detection, allowing for efficient processing of a large number of samples and reliable results with minimal background interference.
How do you conjugate antibodies with HRP?
Antibodies can be conjugated to HRP through chemical crosslinking methods that typically involve modifying functional groups on both the antibody and the enzyme, such as amine or thiol groups, using reagents like glutaraldehyde, periodate, or NHS-ester-based crosslinkers; these modifications create stable, covalent bonds between the antibody and HRP, resulting in an active conjugate suitable for immunoassays.
Which experimental applications can HRP-conjugated antibodies be used for?
HRP-conjugated antibodies are widely used in western blotting (WB), immunoprecipitation (IP), dot blot (Dot), immunomicroscopy (IM), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), and immunohistochemistry on frozen (IHC-Fr) and paraffin-embedded (IHC-P) tissues, enabling sensitive detection and signal amplification across various experimental applications.