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overexpression FLAG-tagged protein shows extra bands in WB

flag overexpression tag

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#1 veronicaalej

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Posted 12 September 2011 - 06:46 AM

Hi,
I've used the system by Stratagene to make N and C-terminal FLAG-tagged proteins and, so far, it worked perfectly. The constructs are perfect. When I checked by WB the expression of the tagged protein in cells transiently transfected with the plasmid I only observed a band corresponding to my protein expected molecular weight (60 kDa). However, when I blotted the membrane with the flag antibody I detected my protein and a few more quite abundant bands of lower molecular weight (around 30 kDa). It seems it might be a matter of degradation of my protein even though the antibody against the protein itself is not recognizing these bands? Now I'm checking the expression pattern in cells stable transfected with the plasmids.
Has anyone ever found these extra bands when overexpressing a FLAG-tagged protein?

Thanks in advance!!

#2 GNANA

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Posted 12 September 2011 - 08:24 AM

it s not the matter of FLAG tag, as you said it might be the degraded protein...may be you confused why only the flag recognises but the ab against the protein doesnt, right?? if that is your concern, it is because the epitope of the ab might have lost in the degraded part of ur protein, to confirm this use an antibody against another epitope..and why FLAG recognizes, because the flag is at the extreme, so where-ever ur protein got sliced the flag epitope will still be hanging at the end...
I would prefer being perfectionist rather than a passionist in Research.

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#3 veronicaalej

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Posted 12 September 2011 - 12:26 PM

Yeah, that was my guess, degradation and I imagine the lack of the epitope due to degradation but the matter that concerns me is why is my protein being degraded?? I've used a different plasmid to overexpress my protein before, also with a flag and flag western blot and I was not seing that pattern of degradation.
Has anybody ever experienced something like this?

I've just done it once so it might be a problem of that particular protein extraction and wb, might be.

Thanks for the reply.





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