Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Multiple Labeling

With BV421™ & BV480™

  • Combine with AF488, RR-X, and AF647 for 5-color imaging
  • Compatible with commonly used filter sets
  • Switch nuclear stain from DAPI to DRAQ5 for additional labeling options

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Whole IgG Affinity-Purified Secondary Antibodies

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Monovalent Fab Fragments for Double Labeling - Example A

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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.

figure 1

figure 1 

  1. Incubate with the first primary antibody,in this example Rabbit Anti-Antigen X. Wash.

  2. Incubate with excess Probe I-conjugated secondary antibody, in this example Alexa Fluor® 488-Fab fragment Goat Anti-Rabbit IgG (H+L). Wash.

  3. Incubate with the second primary antibody, Rabbit Anti-Antigen Y, followed by Probe II-conjugated secondary antibody, in this example Rhodamine Red-X-Goat Anti-Rabbit IgG (H+L). Wash.

Caution: This protocol may be subject to the following problems:

(1) Example A may require a high concentration of conjugated Fab to achieve effective blocking of the first primary antibody. If this results in unacceptable background, try a lower concentration of the conjugated Fab, followed by further blocking with unconjugated Fab.

(2) If small aggregates of conjugated Fab are bound to the first primary antibody, they may act as divalent or polyvalent antibodies and capture some of the second primary antibodies, which would result in overlapping detection of antigens.


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