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I performed immunofluorescence staining with my labeled antibody, but I don’t see any signal. What should I do?

  1. Check with the antibody manufacturer to confirm that the antibody formulation and concentration are compatible with the kit labeling protocol you selected.
  2. You should confirm that your primary antibody is sensitive and specific for your application using indirect labeling before attempting direct labeling. You may need to use a higher concentration of primary antibody to achieve similar signal intensity with direct labeling as with indirect labeling.
  3. Covalent labeling may affect the reactivity of certain antibodies. You can confirm that the labeled antibody is still reactive by performing indirect immunofluorescence labeling with your Mix-n-Stain labeled primary followed by a fluorescently-labeled secondary antibody.
  4. You can confirm labeling of your antibody by performing denaturing SDS-PAGE on a small amount (0.1-0.5 ug) of labeled antibody, then imaging the gel fluorescence at the appropriate excitation/emission wavelengths of the CF® dye you used. You should be able to detect fluorescent bands representing IgG heavy and light chains at ~55 kDa and ~25 kDa.

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