Example B. Use of unconjugated Fab fragments to convert the first, primary antibody into a
different species.
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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 an excess of unconjugated Fab antibody directed against the host species of
the primary antibody. |
3. |
Incubate with Probe 1-conjugated tertiary antibody [an anti-IgG (H+L) or an
anti-F(ab')2] directed against the host species of the Fab antibody. It is very important that the tertiary
antibody does not recognise the host species of the either the primary antibodies or the second, secondary antibody. |
 |
 |
| 4. |
Incubate with the second, primary antibody. |
5. |
Incubate with Probe II-conjugated to the second, secondary antibody (that does not recognise
the host species of either the Fab antibody or the tertiary antibody). |
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. |
