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His-tag purification post Enterokinase digestion


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#1 darel_am

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Posted 27 January 2011 - 03:24 AM

I have a his-tagged fusion protein that has an enterokinase site near the centre so when I cleave, you can't be sure which band is what in the resulting gel (both products run a similar distance). I was planning on running my Ek digestion over a Ni column and the flow through should contain my desired protein and the eluate my His-tagged terminus. However, I am unsure whether I need to exchange my buffers after the Ek digestion step as the column protocol is based on purification post E. coli cell lysis? Has anyone done this and have any pointers before I begin?!

Thanks

Edited by darel_am, 27 January 2011 - 03:25 AM.


#2 vrindasim23

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Posted 08 December 2011 - 09:27 AM

I am also facing a similar trouble. The fusion protein was purified by His tag column. The purified protein was enterokinase digested and run on gel, I am unable to find any difference between the fusion protein and the protein of my interest. Tried purifying further using the His tag column again but the same pattern of protein band is present in the lysate flow through, wash flow through and none in elution buffer. trying to find a solution but none is coming out fine....what should i conclude from this pattern of results....Thank you all for your valuable suggestions if any.....




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