I immunofluresence stained a membrane protein, but after 594 goat-anti-rabbit secondary antibody incubation, I found strong nucleus signal under microscope.
Do you have any idea why this happened? Thanks.
Did you do a secondary-only (no primary) control? If you do this, with the same blocking protocol, and still see the nuclear signal, then this is due to non-specific dye charge binding, where the negatively-charged dye is binding to the positively-charged nuclear proteins. Standard protein blockers won't stop this. Instead, you'd need to use the Image-iT FX Signal Enhancer Solution (sold by Thermo Fisher Scientific as catalog R37107) as an additional blocking step.
You might also be using too high of a concentration or too long of a label step. For cells in culture, I recommend using 5ug/mL of the secondary for 30 minutes at RT.
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