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
