I incubated my pelets in 8M urea for a couple of hrs at 37C, spun and
collected the supernatant, and then refolded the protein over a Ni column,
as I had a his tag protein. To do this, you load and wash the protein
normally, but before eluting, run b-cyclodextrin (i think 0.1%, you'll
have to try a few concentrations) and then a detergent (I used Triton
X-100). Then you can elute. Here's the paper I used as a guide:
Biotechnol Appl Biochem 2006, 43:137-145
Except it's the other way around - the column is washed in the presense of
detergent which is then slowly removed by b-cyclodextrin washes. If the
reversal thing worked for you, your protein likely does not require neither
b-cyclodextrin nor Triton for refolding.
I've had more luck in on-column refolding by steps of decreasing urea
concentrations: 2, 1, 0.5, 0 M (3-5 column volumes each with 30 min in
between at room temperature).
Dima
