Example C. Use of unconjugated Fab fragments for blocking after the first, secondary
antibody step.
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| Key: |
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Rabbit anti-Antigen X |
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Mouse anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) |
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Rabbit anti-Antigen Y |
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Fluorescein (FITC) |
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Fab fragment Goat anti-Rabbit IgG (H+L) |
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Rhodamine Red-X (RRX) |
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Goat anti-Rabbit IgG (H+L) |
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Rabbit IgG from Non-Immunized Hosts |
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Mouse anti-Goat IgG (H+L)(min X Ms, Hu, Rb Sr Prot) |
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| 1. |
Incubate with the first, primary antibody. |
2. |
Incubate with Probe I-conjugated to the secondary antibody. |
3. |
Incubate with normal serum (as a source of non-immune IgG) from the same host species as the primary antibodies. The purpose of this step is to saturate any open antigen binding sites on the first, secondary antibody so that it can not bind the second, primary antibody. |
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| 4. |
Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibody. In this case the Fab antibody should come from the same host species as the conjugated, secondary antibody. |
5. |
Incubate with the second, primary antibody. |
6. |
Incubate with Probe II-conjugated to the same secondary antibody as used
in step 2. |
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. |
