[Protein-analysis] His-tagged protein purification from yeast (k lactis) supernatant

Tiwari via proteins%40net.bio.net (by kstiwari from gmail.com)
Thu Jan 3 00:44:07 EST 2008


Hi Guys,

I am expressing the recombinant protein 6xHis tagged at C' terminal
secreting in YP-Galactose media by K lactis. My problem is whenever i
try to purify by using Ni-NTA column, it turns the column white as if
supernatant is chelating Ni from the column. I tried to condition
supernatant by adjusting pH 8.0 and Salt upto 1M with no success.
Please suggest me what should i do. Ammonium sulphate precipitation
result in inactivation of protein. i cant do dialysis economically to
huge culture volume.  so these will not be a good option for
conditioning media.

Thanks!


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