Horseradish Peroxidase (HRP) Secondary Antibodies

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HRP Conjugated Secondary Antibodies

This commonly used reporter enzyme is derived from the root of the horseradish plant (Armoracia rusticana). JIR HRP conjugates are prepared by a modified Nakane and Kawaoi procedure (1974).

HRP conjugates are suitable for all immunotechniques employing colorimetric and chemiluminescent detection methods, including Western blotting, immunohistochemistry and ELISA. Jackson ImmunoResearch provides a wide range of secondary antibodies, with comprehensive options for host and target species, as well as immunoglobulin specificities.

Horseradish peroxidase is available conjugated to:

• Whole IgG Secondary Antibodies
• F(ab')2 Fragment Secondary Antibodies
• Anti-Fluorescein, Anti-Digoxin, and Anti-Biotin Antibodies
• Streptavidin
• ChromPure™ Purified Proteins from Normal Serums

We also offer Peroxidase-Anti-Peroxidase (PAP) as as an alternative labeling approach.

Western Blotting with JIR Secondary Antibodies Western Blot Troubleshooting Guide

Note about endogenous peroxidase

Some tissues contain endogenous peroxidase-like enzymes which can react with peroxidase substrates, resulting in background staining. Pre-treatment of sample with hydrogen peroxidase will exhaust the endogenous enzyme activity, allowing clear detection of specific signal.

Cautions regarding reagent compatibility:

1. Do not add sodium azide to solutions containing HRP. It will inactivate the enzyme. See Blocking and Controls Guide for details on how to check enzyme and substrate reactivity.
2. If glycerol is added to extend shelf life of reconstituted product, confirm that glycerol is ACS grade or better. Lower grades of glycerol may seriously inhibit peroxidase enzyme activity.

Colorimetric detection

HRP conjugates can be used for colorimetric detection. The conjugated reporter enzyme catalyzes the conversion of the chromogenic substrate to a colored precipitate, visualized directly on the blotting membrane or tissue sample. Colorimetric detection can offer quick and easily obtained results without the need for expensive detectors or extensive optimization.

Read our blog about Colorimetric Western blotting

Chemiluminescent detection

Enzyme-linked conjugates can also be used for chemiluminescent signal detection. HRP conjugates produce signal by oxidizing a chemiluminescent substrate (luminol) to a form which emits light. AP conjugates produce signal when the enzyme dephosphorylates a specific substrate (e.g. 1,2-dioxetane) to a light emitting product. The signal can then be captured by exposing photographic film to the membrane or using a cooled charge-coupled device (CCD) camera. Chemiluminescent detection offers excellent sensitivity, however quantification and probing for multiple targets can be limited, and development may require refinement to optimize signal capture.

Read our blog about Chemiluminescent Western blotting

Specificities Available

• Bovine IgG and Fc fragment specific
• Chicken IgY (IgG), F(ab')2 and Fc fragment specific
• Cat IgG, F(ab')2 and Fc Fragment specific
• Dog IgG and F(ab')2 fragment specific
• Goat IgG, F(ab')2, Fc fragment, and light chain specific
• Guinea Pig IgG, F(ab')2 and Fc specific
• Armenian Hamster IgG
• Syrian Hamster IgG
• Horse IgG and Fc fragment specific
• Human IgA + IgG + IgM
• Human Serum IgA, α Chain Specific
• Human IgG, F(ab')2, Fcγ Fragment and light chain specific
• Human IgM, Fc5μ fragment specific
• Human Lactoferrin
• Mouse IgG, Fcγ fragment specific
• Mouse IgG, Fcγ subclass specific (1 / 2a / 2b / or 3)
• Mouse IgM and µ chain specific
• Rabbit IgG, F(ab')2, Fc and light chain specific
• Rat IgG, Fcγ Fragment specific
• Rat IgM and IgM μ chain specific
• Sheep IgG, F(ab')2, Fc fragment and light chain specific
• Swine IgG
• anti-biotin
• anti-digoxin
• anti-fluorescein

Anti-Horseradish Peroxidase Antibodies

Affinity-purified anti-horseradish peroxidase antibodies are available for detection of horseradish peroxidase antigen, or for signal amplification of HRP-containing reagents. For immunostaining of mammalian cells, an advantage of using anti-horseradish peroxidase is reduced background, since the antibody does not recognize the endogenous peroxidase-like enzymes found in those cells.

Read more about signal enhancement with anti-HRP

References

• Paul K. Nakane, Akira Kawaoi (1974). Peroxidase-labeled Antibody A New Method Of Conjugation Journal of Histochemistry & Cytochemistry Vol 22, Issue 12, pp. 1084 - 1091. 10.1177/22.12.1084

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