Dear Peter,
The method I use for refolding his tagged proteins while bound to the column
is as follows
1. Suspend the cells in 20 mM tris, 50 mM NaH2PO4, 250 mM NaCl,
pH8.0, (Call this buffer A)10 mM imidazole, 50ug/ml lysozyme, 0.5 %
tween 20. I use approx. 5ml buffer per gram of wet cells. Freeze at
-20C overnight or longer.
2. I pass the cells through a french press after thawing,
alternatively you could just pass them through a syringe to shear
the DNA. Also you could just lyse them by sonication (in which case
don't bother freezing unless you want to) it doesn't really matter.
Spin the lysate at 15000g for 30 min, 4C.
3. Suspend the pellet in buffer A with 8 M urea10% glycerol and 10 mM
imidazole spin at 15000g, 30 min 25C. (check the pH because urea and
imidazole change it)
4. Incubate the urea fraction with pre-eqilibrated NTA agarose for 1
hour with gentle shaking at R.T.
5. Put the suspension in a gravity column (we use small column with
about a 1 cm diameter) and remove the unbound material. Wash the
column with 5 volumes of resuspension buffer, 5 volumes buffer B, 5
volumes buffer C, 10 volumes buffer buffer D, 10
volumes buffer E and elute with 5 volumes elution buffer.
B: A plus 5% glycerol, 10 mM imidazole and 6 M urea.
C: A plus " " 3 M urea
D: A plus " " 1 M urea
E: A plus " , 20 mM imidazole and no urea.
Elution: A plus 5% glycerol, 250 mM imidazole.
I have had some problems with one of my proteins not binding well with the
10mM imidazole in the buffer so you might have to remove this if you
experience similar problems
"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
--
Scott Craig
Dept. of Biotechnology
University of New South Wales
Sydney, Australia
