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inespecific bands in nuclear fraction?

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#1 valeri_a



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Posted 17 January 2013 - 03:58 AM

HI all,

I working with a cosntruct linked to GFP, I supposed that this protein could be a transcriptional factor. and for that, after transfection and adecuate treatment, I made a nuclear extraction to see my tagged protein in differents fraction by western blot. I saw very good expression in the cytoplasmic region, but also I saw strong band in the insoluble fraction, but near to 15 kDa. I tryied with antibodies against GFP and my protein, and appeared the same band.
I don´t understand, because GFP has a MW 25 Kda, and for that I supposed found bands higher than 25 Kda.
and because I don`t have too much experience in nuclear extraction, I don´t know if this signal could be an inespecific band, or histones, or anything else.

another thing. could I use lamin A/c as a marker of the soluble nuclear fraction??

THank you for your comments,

See u!

#2 bob1


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Posted 17 January 2013 - 11:55 AM

It is possible that the protein in the insoluble fraction is degradation products. If you have tritrated the antibody so that it gives specific signal for a known GFP positive sample and you used this on your insoluble fraction, then the signal should be specific.

You can use Lamin, but you might be better off using nucleolin, histone H3 or p53 as nuclear markers.

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