Monovalent Fab Fragments for Double Labeling - Example B

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Fab Fragments for Blocking and Double Labeling of Primary Antibodies from the Same Host Species

Example B. Use of unconjugated Fab fragments to cover the first primary antibody, presenting it as a different species.

1. Incubate with the first primary antibody, in this example Rabbit Anti-Antigen X. Wash.


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.


3. Incubate with Probe I-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.


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


5. Incubate with Probe II conjugated to the second 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.

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