HRP Conjugated AffiniPure Rabbit Anti-Human IgG (H+L)

Product Overview

Assay Information

Main Advantages

Background

Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species. The host antiserum is then purified through immunoaffinity chromatography to remove all host serum proteins, except the specific antibody of interest. Purified secondary antibodies are further solid phase adsorbed with other species serum proteins to minimize cross-reactivity in tissue or cell preparations, and are then modified with antibody fragmentation, label conjugation, etc., to generate highly specific reagents. Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, and fluorescent dyes/proteins. Here, the antibody provides the specificity to locate the protein of interest, and the label generates a detectable signal. The label of choice depends upon the experimental application.

Horseradish peroxidase (HRP) is extensively used for labeling secondary antibodies in ELISA, western blot, dot blot and immunohistochemistry. The HRP enzyme is made visible using a substrate that, when oxidized by HRP in the presence of hydrogen peroxide as an oxidizing agent, yields a characteristic change that is detectable by specific detection methods. The substrates commonly used with HRP fall into different categories including chromogenic, fluorogenic, and chemiluminescent substrates depending on whether they produce a colored, fluorimetric or luminescent derivative respectively. The intensity of the signal is proportional to peroxidase activity and is a measure of the number of enzyme molecules reacting, hence of the amount of recognized primary antibodies, and thus of the amount of target antigen.

Assay Dilutions Recommendation

The recommendations below provide a starting point for assay optimization. Actual working concentration varies and should be decided by the user.

Western Blotting: 0.1-0.2μg/ml (ECL detection) Western Blotting: 0.7-3.3μg/ml (DAB detection) ELISA: 0.05-0.5μg/ml (TMB detection)

Validation Images & Assay Conditions

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Boster Kit Box