Jackson Immuno Research Inc.
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Whole IgG Affinity-Purified Secondary Antibodies

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Detection of two unlabeled primary antibodies from the same host species

Example A: Key of Elements.
Example A: Key of Elements.
Use of conjugated Fab fragments for labeling and blocking: 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 conjugated Fab fragments for labeling and blocking: step two.

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

Use of conjugated Fab fragments for labeling and blocking: step three.

Step 3. Incubate with the second primary antibody, Rabbit Anti-Antigen Y.

Use of conjugated Fab fragments for labeling and blocking: step four.

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

Example A: Use of conjugated Fab fragments for labeling and blocking


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 conjugated Fab fragments for labeling and blocking: step one. Use of conjugated Fab fragments for labeling and blocking: step two. Use of conjugated Fab fragments for labeling and blocking: step three. Use of conjugated Fab fragments for labeling and blocking: step four.

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 excess conjugated secondary antibody, in this example Alexa Fluor® 488-Fab fragment Goat Anti-Rabbit IgG (H+L). Wash.

Step 3. Incubate with the second primary antibody, Rabbit Anti-Antigen Y.

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

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Example A: Key of Elements.

Application Notes:

  • Monovalent Fab fragments have not been adsorbed against other species, so they may cross-react with endogenous Ig. Use Example C to avoid detection of endogenous Ig.
  • Example A may require a high concentration of conjugated Fab to saturate the first primary antibody. If this results in unacceptable background, try a lower concentration of the conjugated Fab, followed by further blocking with unconjugated Fab.

Other Fab Blocking Protocols


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