detergent removal and protein precipitation

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Tue May 17 03:36:00 EST 2005


"Martin" wrote:


> 1. I plan to use a mild detergent to solubilize the monomer, subject it
> under disulphide-bond promoting conditions, and remove the detergent
> afterwards. My lab has SDS, Triton-X-100, NP-40 and Tween-20 only. Will
> Tween-20 suit my purpose?

SDS is everything but mild, for most proteins it's strongly denaturing.
The non-ionic detergents are milder. You may also try
cetyltrimethylammonium bromide (CTAB), a positively charged detergent,
highly solubilising but surprisingly mild. Also relatively cheap.
> 
> 2. How to remove Tween-20 from protein sample? I am thinking about using
> acetone (in excess volume) precipitation because tween-20 is soluble in
> acetone. Will this work?

Aceton precipitation is a classical method for protein purification, but
acetone is denaturing on proteins, especially at higher temperatures.
Use the aceton very cold (-20 degrees or less) and add it slowly to the
ice-cold sample as mixing acetone and water produces heat. Spin the
protein precipitate down (at -10 degrees) and lyophilise the pellet to
remove the acetone. Only when all acetone is removed re-dissolve in a
buffer of your choice. For small volumes, methanol/chloroform
precipitation (Wessel & Fluegge) allows better recovery.

Alternatively, use BioBeads (from BioRad) to bind the detergent by
hydrophobic interaction. The protein stays in solution. The following
review (available on the net) should direct you to detailed description
of the method:

@article{Rig-98,
        AUTHOR= {J.L. Rigaud},
        TITLE= {Membrane proteins: functional and structural studies
using reconstituted proteoliposomes and 2-{D} crystals},
        YEAR= {2002},
        JOURNAL= {Braz. J. Med. Biol. Res.},
        PAGES= {753-766},
        VOLUME= {35},
        LANGUAGE= {engl}
}

Pierce offers small disposable columns of a similar material for
detergent removal from proteins.

> 
> 3. What's the usual effect of protein precipitation on protein? What's
> the difference among precipitation reagents?

That depends on the protein. Some proteins are difficult to redissolve,
others survive the procedure quite well. Impossible to predict.





More information about the Methods mailing list