subunit separation

D.K. dk at no.email.thankstospam.net
Tue Nov 26 22:51:13 EST 2002


In article <3DE24661.97428347 at vub.ac.be>, Sigrid Van Boxstael <svboxsta at vub.ac.be> wrote:
>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
>polypeptides.
>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
>supernatans.
>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?
>

Assuming you did everything right, repeat the same but with 6M GuHCl
instead of urea. Can heat up a bit to ensure complete denaturation. 

Since you obviously do not care for the proteins being intact, why not cut
our the appropriate band from the gel? (Zn-imidazole or copper staining, 
both reversible). 

Finally, since these are recombinant proteins, why not express them 
separately?

DK



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