Jackson Immuno Research Inc.
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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 two unlabeled primary antibodies from the same host species

Example C: Key of Elements.
Example C: Key of Elements.
Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step one.

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

Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step two.

Step 2. Incubate with conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.

Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step three.

Step 3. Incubate with normal serum from the same host species as the primary antibodies, in this example normal rabbit serum. The purpose of this step is to saturate open binding sites on the first secondary antibody with IgG so that they cannot capture the second primary antibody. Wash.

Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step four.

Step 4. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibodies, in this example Fab Goat Anti-Rabbit IgG (H+L). The host species of the Fab antibody should be the same as the host species of the conjugated secondary antibody. This step covers the rabbit IgG so that the second secondary antibody will not bind to it. Wash.

Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step five.

Step 5. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step six.

Step 6. Incubate with the same secondary antibody as used in step 2, conjugated to a different probe, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.

Example C: Use of unconjugated Fab fragments for blocking after the first secondary antibody step.


Examples A, B and C show some of the possible protocols used for double labeling two unconjugated primary antibodies from the same host species.

The success of these experimental designs will require some empirical manipulations. Optimizing reagent concentrations in each step or switching the labeling sequence of the two antigens may influence the outcome.

  • Labeling the less abundant primary antibody first increases blocking efficiency.
  • Blocking with an appropriate normal serum helps to reduce background.
  • To avoid displacement of the Fab antibody by the labeled secondary antibody, a light post-fixation with glutaraldehyde may be used, provided that it does not affect antigenicity of the target proteins.
Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step one. Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step two. Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step three. Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step four. Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step five. Use of unconjugated Fab fragments for blocking after the first secondary antibody step: step six.

Step 1. After blocking with normal serum, incubate with the first 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) (min X Hu, Ms, Rat Sr Prot). Wash.

Step 3. Incubate with normal serum from the same host species as the primary antibodies, in this example normal rabbit serum. The purpose of this step is to saturate open binding sites on the first secondary antibody with IgG so that they cannot capture the second primary antibody. Wash.

Step 4. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibodies, in this example Fab Goat Anti-Rabbit IgG (H+L). The host species of the Fab antibody should be the same as the host species of the conjugated secondary antibody. This step covers the rabbit IgG so that the second secondary antibody will not bind to it. Wash.

Step 5. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Step 6. Incubate with the same secondary antibody as used in step 2, conjugated to a different probe, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.

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Example C: Key of Elements.

Other Fab Blocking Protocols


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