detergent removal help!

Shaun D. Black SHAUN at JASON.UTHCT.EDU
Wed Jun 22 21:54:15 EST 1994


scott_basehore at stratagene.com wrote on 6/22/94:
> 
>           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"
> 
First, dialysis really will work, but because you are well above the cmc of
the detergents, the dialysis process will takes a couple of days instead of
a couple of hours for typical non-micellar molecules.  NP-40 has useful
absorbance at 280nm.  As you dialyze, read A-280nm until it comes to 
equilibrium.  Repeat dialyses until soap is reduced to an appropriate level.

Second, a straightforward (and far faster) method to remove detergent is
to bind your protein to a column (e.g. ion exchange or hydroxylapetite) and
wash the column with detergent-free buffer until the A-280nm drops to zero.
Then, elute your protein with the appropriate buffer.

I hope this helps!  Best regards,  Shaun   (shaun at jason.uthct.edu)



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