Refolding His-tagged proteins

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Jun 29 05:35:56 EST 1999


One easy method worth a try is to
- prepare inclusion bodies by sonication
- wash them thoroughly in your sonication buffer (or plain water)
- dissolve in a small volume of 0.5 to 1% SDS in a buffer of your choice
- rapidly dilute in a large volume so that the SDS concentration is <0.05%
- add Triton X-100 to 1% final concentration
- let stand for 5min at RT
- spin down insoluble matter for 10min at 10000g (there may be no pellet:
good sign)
- use supernatant to perform purification over Ni-column as if you had
soluble protein

Hope this helps,
Frank


"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




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