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Seeing two SDS-PAGE bands upon protein expression from pET-30b(+)


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#1 Zaineb

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Posted 29 March 2011 - 05:36 AM

Hi,
So I expressed my protein in E. coli and then purified it using his-tag affinity purification. When I run the purified protein on SDS-page I see two bands! One corresponds to my protein but the other one is a couple of kD's higher in MW compared to my protein. I don't think it is a contamination of another protein because this band does not exist in my crude extract sample before induction. And when I run my crude extract after induction I see this band which means it's something that gets expressed and induced after induction but i am not sure what it is.
The vector I used is pET-30b(+).
Any help will be appreciated =).

#2 protolder

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Posted 29 March 2011 - 09:50 PM

Hi,
So I expressed my protein in E. coli and then purified it using his-tag affinity purification. When I run the purified protein on SDS-page I see two bands! One corresponds to my protein but the other one is a couple of kD's higher in MW compared to my protein. I don't think it is a contamination of another protein because this band does not exist in my crude extract sample before induction. And when I run my crude extract after induction I see this band which means it's something that gets expressed and induced after induction but i am not sure what it is.
The vector I used is pET-30b(+).
Any help will be appreciated =).

Hola, I have seen the plasmid map and it is of constitutive expression so you donīt need induce if only want to have your band. Have you purified your extract in a column with an imidazol gradient? how many peaks you see? because multimers elute later.Is your loadong buffer fresh?, add 1ul of mercapto ethanol to your samples and see if the big band disappears that will mean that you have mediated -S-S- dimers. Buena suerte




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