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 Mouse anti-Rabbit IgG (H+L) Mouse anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot)
Rabbit anti-Antigen Y Rabbit anti-Antigen Y Fluorescein Fluorescein (FITC)
Fab fragment Goat anti-Rabbit IgG (H+L) Fab fragment Goat anti-Rabbit IgG (H+L) Lissamine Rhodamine Rhodamine Red-X (RRX)
Goat anti-Rabbit IgG (H+L) Goat anti-Rabbit IgG (H+L) Rabbit IgG from Non-Immunized Hosts Rabbit IgG from Non-Immunized Hosts
Mouse anti-Goat IgG (H+L) Mouse anti-Goat IgG (H+L)(min X Ms, Hu, Rb Sr Prot)


1 2 3

Caution:  This protocol may be subject to the following problems: 1.) Example A may require a higher concentration of conjugated Fab to achieve effective blocking of the first primary antibody. If this results in a higher background, try a lower concentraton of the conjugated Fab followed by further blocking with unconjugated Fab. 2.) If small aggregates of conjugated Fab are present and they bind to the first primary antibody, they may also act as divalent or polyvalent molecules and capture some of the second primary antibody, which could result in overlapping detection of antigens.

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.


 
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