Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates
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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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Peroxidase-Anti-Peroxidase (PAP) Soluble Immune Complexes

PAP soluble immune complexes are prepared by the method of Sternberger et al. (J Histochem. Cytochem. 1970. 12, 315). Theoretically, they consist predominantly of two anti-HRP antibodies in soluble complex with three molecules of HRP.

PAP soluble complexes are normally used at 25-50 μg/ml. Whole antisera against IgG (H+L) are recommended for bridging PAP to primary antibodies. Antisera against IgG (H+L) or against the F(ab')2 fragments of IgG will also bridge PAP to IgM primary antibodies (such as IgM monoclonal antibodies) by virtue of common light-chain recognition. Normal serum (5% v/v) from the same host species as the bridging antibody is suggested as a blocking solution to minimize non-specific binding. All PAP soluble complexes are freeze-dried at 20 mg/ml.

Endogenous peroxidase activity: PAP soluble complexes, as well as other immunoperoxidase reagents, are not recommended for tissues or cells in which endogenous peroxidase activity is difficult to suppress. In such cases, other immunoenzyme reagents may be used. Alternatively, anti-horseradish peroxidase conjugated with other enzymes or fluorophores may be used to enhance signal and reduce background, since the final signal does not depend on the enzyme activity of peroxidase, but on the antigenicity of horseradish peroxidase.

Steps of the PAP Method
A. Primary antibody is bound to antigen, and a bridging antibody has been added to link the primary antibody with corresponding PAP complex.
B. PAP complex (three molecules of HRP complexed with two molecules of anti-HRP) has been captured by bridging antibody.