Sigrid Van Boxstael
svboxsta at vub.ac.be
Mon Nov 25 10:48:49 EST 2002
I am studying a protein which is a dodecamer, composed of two different
polypeptitdes one assembling into trimers and the other one into dimers:
so the complete protein consists out of two trimers of one polypeptide
(the catalytic subunit) and three dimers of the other (the regulatory
subunit). At the N-terminus of the catalytic polypeptide there is a
His-tag linked. By using a Ni-column, it was possible to purify the
protein. Now I am interested in separating the regulatory subunit from
the catalytic subunit.
I thought the following would be a good idea :
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)
As possibilites I see
1) The protein did not unfold and everything is attached at the Ni-resin
2) The regulatory precipitated in the presence of urea?
Does anybody has any comments or suggestions to solve this problem or
maybey a much easier way to separate both subunits?
Can a protein precipitate in such a huge amount of urea?
Thanks in advance,
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