Jackson ImmunoResearch Europe, Ltd

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

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


Key:
	Rabbit anti-Antigen X	Mouse anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot)
	Rabbit anti-Antigen Y	DyLight 488
	Fab fragment Goat anti-Rabbit IgG (H+L)	Rhodamine Red-X
	Mouse anti-Goat IgG (H+L)(min X Ms, Hu, Rb Sr Prot)	Rabbit IgG from normal rabbit serum

Incubate with the first primary antibody, in this example rabbit anti-antigen X. Wash.

Incubate with an excess of unconjugated Fab anti-body against the host species of the primary antibody, in this example unconjugated Fab frag-ment goat anti-rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash.

Incubate with Probe 1-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, in this example DyLight 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, and Shp Sr Prot) was used. Wash.

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

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

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.