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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