detergent removal help!

Warren Gallin wgallin at gpu.srv.ualberta.ca
Wed Jun 22 19:24:46 EST 1994


In Article <9405227723.AA772327701 at strata.stratagene.com>,
scott_basehore at stratagene.com wrote:
>
>          I have purified an enzyme in a buffer with 0.1% NP-40 and
>          0.1% Twn-20.  In order to grow crystals of my enzyme,
>          suitable for X-ray crystallography, I need to remove these
>          two detergents. Dialysis won't work as the detergents are
>          above the critical conc. for the formation of micelles. I
>          have tried an Extracti-gel D (Pierce) column, but the
>          solution still foams when agitated (low-tech assay).  I am
>          fairly sure that my enzyme alone will not foam at its
>          present concentration.  Does anyone know a method for
>          removing these detergents?  How might one assay for the
>          presence of detergents?
>                       "I am approaching the hypotenuse of my angst"

We use SM-2 BioBeads, from BioRad.  They are a large, porous bead that
absorbs Triton X-100 like crazy (NP-40 = Triton X-100)  You simply incubate
the solution with some biobeads for an hour or so, then filter the beads out
through a plug of glass wool, and presto!, detergent-free (or nearly so)
solution.  You might want to dialyse at this point to get rid of the
detergent remaining as monomers.  As far as monitoring, NP-40 has a huge
absorbance at 280 nm, one reason why you can't use A280 to monitor column
effluent if the detergent is present.  Try running an absorbance spectrum
and you should be able to tell when the detergent is gone and only the
enzyme is left.


Warren Gallin,
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca



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