Dr Engelbert Buxbaum
engelbert_buxbaum at web.de
Fri Nov 29 08:13:40 EST 2002
Sigrid Van Boxstael wrote:
> 1)Dialysis of the protein (95 % pure) against 8 M urea, 0.1 M tris pH
> 8.2, 10 mM mercapto-ethanol; Purpose : unfolding of the protein so I
> get free his-linked catalytic polypeptides and free regulatory
> This dialysis happenend during two hours at room temperature.
> 2) Putting the dialysed protein on the Ni-resin. Purpose : The
> catalytic polypeptides stays on the resin, the regulatory will be in the
> 3) Putting the supernatans on SDS-gel to check.
> What do I see on the SDS-gel with the supernatans : Nothing.
> (The gel and staining were OK, the dialyzed protein shows both bands)
Try gel chromatography (with urea in the running buffer). If you have
incomplete denaturation, you should get one peak at the MW of the
dodecamere, otherwise two peaks at the MW of regulatory and catalytic
Note that if the subunits are crosslinked by disulfide bridges,
inclusion of bME alone may not break these bonds. You may have to
incubate for 1 h at 37 degrees, or even boil (shudder).
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