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 an excess of unconjugated Fab antibody against the host species of the primary antibody, in this example unconjugated Fab fragment Goat Anti-Rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash.
Step 3. Incubate with conjugated tertiary antibody directed against the host species of the Fab antibody. The tertiary antibody must not recognize the host species of either the primary antibodies or the second secondary antibody. This example used Alexa Fluor® 488-Mouse Anti-Goat IgG (H+L) (min X Ms, Hu, Rb Sr Prot). Wash.
Step 4. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.
Step 5. Incubate with second conjugated secondary antibody, that does not recognize the host species of either the Fab antibody used in step 2 or the tertiary antibody used in step 3. In this example, Rhodamine Red™‑X-Mouse Anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) was used. Wash.
Example B: Use of unconjugated Fab fragments to cover the first primary antibody, presenting it as a different species
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.
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 an excess of unconjugated Fab antibody against the host species of the primary antibody, in this example unconjugated Fab fragment Goat Anti-Rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash.
Step 3. Incubate with conjugated tertiary antibody directed against the host species of the Fab antibody. The tertiary antibody must not recognize the host species of either the primary antibodies or the second secondary antibody. This example used Alexa Fluor® 488-Mouse Anti-Goat IgG (H+L) (min X Ms, Hu, Rb Sr Prot). Wash.
Step 4. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.
Step 5. Incubate with second conjugated secondary antibody, that does not recognize the host species of either the Fab antibody used in step 2 or the tertiary antibody used in step 3. In this example, Rhodamine Red™‑X-Mouse Anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) was used. Wash.
Application Note:
- 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.
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 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.
Read More
