Example D. Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies.
If it is necessary to incubate with the labeled primary antibody first, then it is essential to block with a monovalent Fab antibody, as shown below, to prevent the labeled secondary antibody from binding to the labeled primary antibody.
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Incubate with Probe I-conjugated to the first primary antibody, in this example DyLight 488-rabbit anti-antigen X. Wash. |
2. |
Incubate with an excess of unconjugated Fab goat anti-rabbit IgG (H+L). Wash. |
3. |
Incubate with the unconju-gated primary antibody, in this example rabbit anti-antigen Y. Wash. |
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Incubate with Probe II-conjugated secondary antibody, in this example Rhodamine Red-X-goat anti-rabbit IgG (H+L)(min X Hu, Ms, Rat Sr Prot). Wash. |
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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 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 E. Detection of one unlabeled and one or more labeled primary antibodies from the same host species without the use of Fab fragments. |
