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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Detection of one unlabeled and one or more labeled primary antibodies from the same host species

Example E: Key of Elements.
Example E: Key of Elements.
Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step one.

Step 1. After blocking with normal serum, incubate with the unlabeled primary antibody, in this example Rabbit Anti-Antigen X. Wash.

Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step two.

Step 2. Incubate with conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L). Wash.

Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step three.

Step 3. Incubate with normal serum from the host species of the primary antibody, in this example normal rabbit serum. Wash.

Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step four.

Step 4. Incubate with conjugated primary antibody, in this example Rhodamine Red™‑X-Rabbit Anti-Antigen Y. Wash.

Example E: Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments


Examples D and E illustrate multiple labeling protocols that include a directly labeled and an unlabeled primary antibody from the same host species. It is advisable to incubate the less abundant primary first. In Example D, the directly labeled primary antibody is incubated first, then blocked with Fab fragments prior to applying the unlabeled primary antibody.

If the unlabeled primary antibody is incubated first (Example E), double labeling can be achieved without using Fab fragments. Following incubation with the labeled secondary antibody, normal serum is used to block open binding arms of the secondary, preventing capture of the labeled primary.

Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step one. Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step two. Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step three. Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments: step four.

Step 1. After blocking with normal serum, incubate with the unlabeled primary antibody, in this example Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L). Wash.

Step 3. Incubate with normal serum from the host species of the primary antibody, in this example normal rabbit serum. Wash.

Step 4. Incubate with conjugated primary antibody, in this example Rhodamine Red™‑X-Rabbit Anti-Antigen Y. Wash.

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Example E: Key of Elements.

Other Fab Blocking Protocols


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