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

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


Key:
Rabbit anti-Antigen X Rabbit anti-Antigen X Goat anti-Rabbit IgG (H+L) Goat anti-Rabbit IgG (H+L)
Rabbit anti-Antigen Y Rabbit anti-Antigen Y Fluorescein DyLight 488
Fab fragment Goat anti-Rabbit IgG (H+L) Fab fragment Goat anti-Rabbit IgG (H+L) Lissamine Rhodamine Rhodamine Red-X


ex-a-1 ex-a-2 ex-a-3
1.
Incubate with the first primary antibody, in this example rabbit anti-antigen X. Wash.
       2.
Incubate with excess Probe I-Fab antibody against the host species of the primary antibody, in this example DyLight 488-Fab fragment goat anti-rabbit IgG (H+L). Wash.
    3.
Incubate with the second primary antibody, rabbit anti-antigen Y, followed by Probe II-conjugated divalent secondary antibody, In this example Rhodamine Red-X-goat anti-rabbit IgG (H+L). Wash.

Caution:  This protocol is subject to the following potential problems: 1.) Example A may require a high concentration of conjugated Fab to achieve effective blocking of the first primary antibody. If this results in unacceptable background, try a lower concentraton of the conjugated Fab followed by further blocking with unconjugated Fab. 2.) If aggregates of conjugated Fab are present and they bind to the first primary antibody, they may act as divalent or polyvalent molecules and capture some of the second primary antibody. This would result in apparent detection of overlapping antigens, even though the antigens may not be overlapping.

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

Example B. Use of unconjugated Fab fragments to convert the first, primary antibody into a different species.

Example C. Use of unconjugated Fab fragments for blocking after the first, secondary antibody step.

Example D. Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments.

Example E. Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies.

NEWS!
   

          Jackson ImmunoResearch now offers selected Alexa Fluor® dye conjugates

 
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