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Why is there extra band in IMAC SDS-PAGE


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#1 chemidiot

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Posted 25 February 2010 - 01:36 AM

Hello friends,

I am running at SDS PAGE on a His-tag purified protein at around 14kDa.

I ran the SDS-PAGE 3 x and each of them have an extra band at the 20kDa.

The SEC peak show only 1 peak and the weight calculated not to be twice as much, so most probable it is not a dimer.

What could be in the 20kDa band? The crude E.coli lysate also display this band.

Can anyone advise??

#2 CambridgeBiochemist

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Posted 28 February 2010 - 04:10 AM

Perhaps you can gel run the cell lysate w/o your plasmid to act as a negative CTRL. This will at least tell you whether that extra band is due to your protein or some other protein from the cells.

Note: many people have reported formation of dimers upon boiling before loading, although your new band is bit lighter to be a dimer.




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