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"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
For colorimetric or chemiluminescent detection Jackson ImmunoResearch offer two reporter enzyme secondary antibody conjugates - horseradish peroxidase (HRP) and alkaline phosphatase (AP). They are suitable for a range of immunoassays, e.g. Western blot, ELISA, IHC and EM.
Jackson ImmunoResearch affinity purified secondary antibodies are used in the detection of an analyte of interest. This can be in solution or when immobilized on solid matrix or tissue. A primary antibody is used to detect the analyte antigen, in turn the primary antibody is recognized by the secondary antibody. Analyte detection is facilitated by using reporter (probe) molecules conjugated to the secondary antibody. The reporter molecules enable visualization of the analyte using either colorimetric, chemiluminescent or fluorescent detection. In colorimetric or chemiluminescent immunoassays the reporter enzyme catalyzes the conversion of a substrate to either a colored or a light emitting product, enabling the detection of the analyte.
JIR Horseradish peroxidase conjugates are prepared by a modified Nakane and Kawaoi procedure (J. Histochem. Cytochem. 1974. 22, 1084). They are suitable for all immunotechniques employing colorimetric and chemiluminescent detection methods, including Western blotting, immunohistochemistry, ELISA and Electron Microscopy.
Figure 2: A colorimetric Western blot. Heavy (HC 50 kDa) and light (LC 25 kDa) chains of reduced and SDS-denatured mouse IgG separated by SDS-PAGE and detected by Western blot using Peroxidase-Goat anti-Mouse IgG (H+L) and visualized with TMB chromogenic substrate.
Alkaline Phosphatase (from calf intestine) conjugates are prepared by a modified method of Avremeas et al., Scand. J. Immunol. 1978. 8 (Supple. 7), 7. The resulting conjugates contain heterogeneous, high molecular weight complexes. They are sensitive reagents for solid-phase immunoassays such as ELISA and Western blotting. Although alkaline phosphatase conjugates are sometimes used for immunohistochemistry, penetration into whole mount tissues may be limited by their large sizes.
Horseradish peroxidase and alkaline phosphatase conjugated secondary antibodies are available in both whole IgG and F(ab') 2 fragment formats.
Available Target Specificities:
Affinity-purified anti-horseradish peroxidase and conjugates 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.