Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Secondary Antibodies

For VHH Discovery

Anti-Alpaca IgG, Subclass and VHH domain secondaries
  • Ideally time PBMC harvest
  • Enhance detection of VHH antibodies
  • Test for VHH expression and binding activity

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Whole IgG Affinity-Purified Secondary Antibodies

"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."

Janet Duerr, Ohio University

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Reporter Enzymes - Horseradish Peroxidase and Alkaline Phosphatase

Secondary Antibody Reporter Enzyme Conjugates

Reporter enzyme-conjugated secondary antibodies can be used for both chromogenic and chemiluminescent detection methods when combined with an appropriate substrate. Enzyme conjugates can be applied to many immunotechniques to provide robust and sensitive detection.

JIR offers secondary antibodies and other reagents conjugated to horseradish peroxidase and alkaline phosphatase.


Horseradish Peroxidase (HRP)

HRP Ribbon Diagram Horseradish peroxidase crystal structure at 1.57 Å from PDB 1HCH. Images generated using Molsoft ICM browser. Image shows two calcium molecules (grey spheres) and the catalytic iron (orange sphere) within the protoporphyrin IX container.

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:

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

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.

Alkaline Phosphatase

Alkaline phosphatase (from calf intestine) conjugates are prepared by a method modified from Avremeas et al., (1978). The resulting conjugates contain heterogeneous, high molecular weight complexes. They are sensitive reagents suitable for solid-phase immunoassays such as ELISA and Western blotting. Although alkaline phosphatase conjugates are sometimes used for immunohistochemistry, penetration into tissues may be limited by their large sizes.

Alkaline phosphatase is available conjugated to:

Alkaline Phosphatase Ribbon Diagram The alkaline phosphatase homodimer from the 2 Å crystal structure PDB 1ALK showing phosphate, zinc and magnesium ions. The image was generated using Molsoft ICM browser.

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

Avremeas, S., Ternynck, T. And Guesdon, J.-l. (1978), Coupling Of Enzymes To Antibodies And Antigens. Scandinavian Journal Of Immunology, 8: 7–23. Doi:10.1111/J.1365-3083.1978.Tb03880.X

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