At 05:23 PM 9/18/2000 +0100, J.betley wrote:
> Dear All, If this is a no-no, then are there any suggestions as to
>alternatives? Thanks Dirk
Dirk,
In general, C18RP can be used effectively to remove these denaturants from
protein solutions (commonly referred to as "desalting" via RP as opposed to
via gel filtration). These chemicals per se will not muck-up your column
but be sure to perform a long enough "wash" to let the salts clear from the
column before eluting your protein. Thats the good news.
Because the protein was denatured, (reduced also???) you likely have
exposed buried hydrophobic groups which were solvated by the denaturants
but now are in an enegetically unfavorable environment. These residues
"prefer"
to interact with "like" i.e., themselves or in your case the C18 functional
groups of the column. If the binding to the column is VERY strong you may
never get your protein to elute. On the other hand, if there is favorable
protein-protein interaction (aggregation???) you may ppt.-out the protein
which may result in a pressure increase. Before trying the desalting on
the column I would simply dilute the denaturant concentration to < 1M or
less, centrifuge the solution, and assay the sup and ppt? for protein.
This should give you some idea of the behavior of the protein minus the
denaturant. A priori, you really don't know what will happen to the sample
following RP desalting. Sometimes you just bite-the-bullet and shoot!
Good luck,
Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-454-5208
e-mail: crimmins@labmed.wustl.edu
This archive was generated by hypermail 2b29 : Wed Sep 20 2000 - 11:12:13 EDT