No signal for immunofluorescence staining w/labeled antibody
Posted: Tue 20 Jan 2015 16:00I performed immunofluorescence staining with my labeled antibody, but I don’t see any signal. What should I do?
FAQ from Biotium
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.
FAQ from Biotium
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.