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Non-specific signal in nucleus

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#1 Obelisk



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Posted 23 June 2009 - 08:11 PM

Dear Sir/Mrs
I have a problem about immunofluorescence staining. I used primary antibody that designed for binding to cytosolic protein but the result showed strong fluorescence signal in nucleus. I think it is non-specific signal. I would like to know this problem can be happened commonly? How could I do to resolve this problem?

Thanks you so much

#2 bob1


    Thelymitra pulchella

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Posted 24 June 2009 - 04:28 PM

Is it truly nuclear staining or just strong peri-nuclear - try confocal microscopy to determine properly? My protein of interest strongly peri-nuclear, and under an ordinary microscope it appears to stain the nucleus, but is not actually in the nucleus.

It is also common for antibodies to stain more than one protein (nonspecific staining) - it may well be that the antibody you are using does this. You can check on a western blot.

You can try changing the conditions for your ICC/IHC - shorter primary incubation, higher dilution of primary, more stringent washes etc.

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