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Detection of two unlabeled primary antibodies from the same host species

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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.

Browse Monovalent Fab Fragments

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.

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

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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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Other Fab Blocking Protocols

Example A

Labeling with two unconjugated primary antibodies of the same host species.

A detection method using conjugated Fab fragments and whole IgG secondary antibodies.

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 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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