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His purification of a regular expressed protein


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#1 beethovenmusic

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Posted 10 March 2003 - 06:22 PM

Hello, does anybody here have experience in purification a his-tagged protein under control of its natural promoter? Do we need to get rid of many other proteins by other techniques (like size exclusion chromatography) before we can mix the cell lysate with the Ni-NTA? Also is it possible to use same Ni-NTA to absorb different batches of cell lysate so that we can use much less amount of Ni-NTA?

#2 volkandemir

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Posted 11 November 2003 - 07:41 PM

I did not understand where your his tag is, I am likely hearing that of course fused to my protein but if your gene is cloned with its native promoter and if your gene has a stop codon in it then where did you fused the histag. If I misunderstood you and your protein is really histagged then you can just follow quigen's Ni-NTA purification protocol... We used nickel agarose protocol (differently from Ni-NTA we used centrifugation instead of coloumn) and used whole cell lyste, but we isolate our protein in denatured form as a single band hence we denatured our protein. Denatured because we did not achived native protein purification using HisTag oloumn, may be the tag was stuck int? the protein...




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