Refolding His-tagged proteins

Rich Dudley rdudley+ at
Tue Jun 29 07:39:43 EST 1999

"Peter S. Galatin" wrote:

> My protein of interest is produced in an insoluble state, so I use
> Novagen's protocol for nickel column chromatography under denaturing
> conditions (I use 6M urea). I can easily elute "plenty" of protein in a
> urea-containing solution, but my attempts to dialyze off/dilute down the
> protein has been met with precipitation. I know that refolding
> conditions are mostly empirically-determined, but could someone direct
> me to a good source discussing the experimental "issues" of refolding
> and maybe something approaching a "flowchart" of things to try... I'd
> love to be able to bind the protein to the column in full 6M urea, and
> progressively step it down before I elute the protein off the beads...
> Thanks for any help,
> Peter

You can find much advice on this in the newsgroup archives at


--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
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