James,
Dima is right. One of the protocols for protein-refolding with
sarcosyl uses cyclodextrins to remove it. However, sarcosyl is an
anionic detergent above pH 5.5 under most conditions, so any ion
exchange platform should work well to markedly reduce its presence in
a preparation. Also, it is extremely insoluble in the presence of
divalent cations (as SDS is) and crystallizes readily. I probably
wouldn't try removing it with an IMAC column as it will bind to some
extent and lead to continued contamination (something we have seen).
It is possible that hydroxyapatite column might be a good matrix for
removing it.
As sarcosyl is anionic, it is slower to dialyze across most membranes.
However, we have had good results for detergent removal just using a
standard spin-desalting column with G-25 Sephedex. It is not
absolutely quantitative detergent removal, but 100x decreases are
easily reached in less than 20 min. For sarcosyl, you could set up
the column with G-25 DEAE-Sephedex at a pH where your protein doesn't
bind and comes out in the flow-through. After 1-2 centrifugations,
your protein should be free of sarcosyl.
Regards,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [email protected]
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On Aug 28, 2009, at 1:24 AM, James Stroud wrote:
Hello All,
I dug around on the net and found this method to remove lipids from
proteins:
"Wessel & Fluegge (1984), Anal. Biochem. 138:141-143. It´s a
methanol/chloroform precipitation and gives you a pellet that is
easily redissolved. The method was especially devised for removing
lipids or detergents, so it should be perfect for you."
-- http://www.bio.net/bionet/mm/methods/1996-December/052513.html
Is this still the preferred way? I do not want to use reagents that
are *themselves* likely to denature my protein. Has anyone tried
cyclodextrins? I'm specifically trying to strip sarcosyl. I want to
do it completely.
James
