Hydrophobic Interaction Chromatography

[H.V.Taylor]

"WB Lai" <wbl20 at cam.ac.uk> wrote:

>I am trying a difficult purification of a denatured protein (suspended in 8
>M urea) on a HIC column.  Has anyone had any success with HIC purification
>in the presence of urea?
>My main problem is that the protein aggregates severely in chaotrope
>concentration < 5M urea.
>Any help is greatly appreciated.
>
What type of HIC are you using? If it's C4-, C8- or phenyl- you could
try adding organic solvents to elute (your protein is denatured
anyway). I'd start with glycerol or ethylene glycol as they will more
likely be compatible with >5M urea than other solvents. However, the
self-aggregation of your protein may be suppressed by glycerol or
ethylene glycol as well. As usual, it's a question of trial and
error... 

Harold