Renaturation of proteins on Ni-NTA column

Hi there,

Has anybody tried to renature proteins bound to Ni-NTA (nickel-agarose)
column? I use Qiagen protocol (washing the resign by linear gradent of
urea in pH=8,0 buffer from 6M to 0,75M). However protein is not folded
(CD,HPLC) moreover it dissapeared stored in 20% glycerol buffer (used
when protein is eluted by 250mM imidasole) and precipitate when try to
dialyse it (eg with GSSG.GSH redox system).
Has anybody had similar problems??



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