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."

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

Example D: Key of Elements.
Example D: Key of Elements.
Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step one.

Step 1. After blocking with normal serum, incubate with conjugated primary antibody, in this example Alexa Fluor® 488-Rabbit Anti-Antigen X. Wash.

Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step two.

Step 2. Incubate with an excess of unconjugated Fab Goat Anti-Rabbit IgG (H+L). Wash.

Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step three.

Step 3. Incubate with the unconjugated primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step four.

Step 4. Incubate with conjugated secondary antibody, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L). Wash.

Example D: Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies


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.

Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step one. Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step two. Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step three. Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies: step four.

Step 1. After blocking with normal serum, incubate with conjugated primary antibody, in this example Alexa Fluor® 488-Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with an excess of unconjugated Fab Goat Anti-Rabbit IgG (H+L). Wash.

Step 3. Incubate with the unconjugated primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Step 4. Incubate with conjugated secondary antibody, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L). Wash.

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Example D: Key of Elements.

Other Fab Blocking Protocols


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