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