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Interpreting a gel


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3 replies to this topic

#1 xsunnysmilesx

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Posted 02 December 2009 - 07:11 AM

I have taken a FLAG-tagged protein and associated it with another protein. When I ran it on an SDS gel and probed the western blot with anti-FLAG antiserum I got 2 bands. I was under the impression that the SDS would denature the proteins so they would dissociate, leaving one band on the western blot? Where could this second band have come from?

Edited by xsunnysmilesx, 02 December 2009 - 07:12 AM.


#2 Vini

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Posted 02 December 2009 - 07:20 AM

I have taken a FLAG-tagged protein and associated it with another protein. When I ran it on an SDS gel and probed the western blot with anti-FLAG antiserum I got 2 bands. I was under the impression that the SDS would denature the proteins so they would dissociate, leaving one band on the western blot? Where could this second band have come from?



whats the size of the bands and that of your proteins? Is it possible that one of the bands actually corresponds to the cleaved flag tag? And what sort of association do the 2 proteins in question have??

#3 laurequillo

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Posted 04 December 2009 - 03:38 AM

It could be an unespecific band, a sumoylation band, a phosphorylation band...it depends of the size of the bands, the buffer you used to do the lysis...maybe you can give us more information, or a picture
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#4 labmed

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Posted 14 December 2009 - 05:35 AM

hi there
I am new to this forum but hoping to get lots of help form you all.
I am facing a very strange problem with my SDS-PAGE. I am cloning my gene in E.Coli (BL21*DE3) strain. Expected protein size is 26KDa.
I have lysed E.coli cell with Lysis Buffer containing imidazol, triton-X 100, glyecrol etc. and protease inhibitor. It was then boiled and freezed repeatedly. After centrifugation the supernatant was treated with loading buffer containing beta mercaptoethanol, bromophenol blue, gylcerol etc. and bolied again for 5 min and then loaded onto 15% gel.
the strange thing is that, after staining with CBBR, i am not able to see any band on the gel, except prestained ladder and when i keep the gel in water (after complete destaining) then after 1 day (kept in water), the bands gets visible and shows the expression of my protein (very clear bands). ;)
what is this happening???????
this doesn't happen when i treat my samples only with loading dye. As soon as i keep the gel in destain for 5 min, faint bands are visible.
is there any problem with my lysis buffer????????? :(
please help me out !!!!

Edited by labmed, 14 December 2009 - 05:38 AM.





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