Polyclonal AffiniPure-VHH™ Secondaries

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New VHH Fragment Antibodies for High-Resolution Immunostaining

Whole IgG Affinity-Purified Secondary Antibodies

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Detection of two unlabeled primary antibodies from the same host species

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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.

Browse Monovalent Fab Fragments

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.

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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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.

Protocol Builder

Primary Antibody Host

Primary Antibody 1 Target

Primary Antibody 2 Target

Secondary Antibody Host

Secondary Antibody 1 Probe

Secondary Antibody 2 Probe

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Step 2.	-
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Other Fab Blocking Protocols

Example B

Labeling with two unconjugated primary antibodies of the same species.

A blocking method using unconjugated fab fragments to “change” the species of one antibody.

Example C

Labeling using two unconjugated primary antibodies of the same species after blocking for endogenous Ig.

A blocking method to allow use of two same species primary antibodies following blocking for endogenous Ig with unconjugated fab fragments.

Example D

Labeling using unconjugated and conjugated primary antibodies of the same host species.

A blocking method using unconjugated fab fragments to prevent detection of the conjugated primary by subsequent antibodies.

Example E

Labeling using conjugated and unconjugated primary antibodies of the same species.

A blocking method using whole Ig antibodies to prevent non-specific labeling.

Other Ways To Use Fab Fragments

Endogenous Blocking

Background staining may be observed if a labeled secondary antibody is not adsorbed to minimize recognition of endogenous tissue Ig.
View Blocking Protocol

FabuLight™ Primary Antibody Labeling

FabuLight antibodies are Fab fragment secondary antibodies specific to the Fc region of IgG or IgM primary antibodies.
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