detergent removal and protein precipitation

Martin sylau at ust.hk
Sat May 14 04:25:29 EST 2005


Hi all,

I am purifying a diffusive signaling ligand from E. coli BL21(DE3)
lysate. According to the literature, its monomer is hardly soluble in
aqueous solvent (my result is consistent). Crystal structure predicts it
to be soluble upon disulphide bond-involved dimerization. I would like
to seek your advice on the following questions or to my work in general:

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?

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?

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

4. I am considering to use a E. coli strain that allows disulphide-bond
formation in cytoplasm. Is the Origami(TM) series from Novagen my only
choice? Can you share your experience in these strains or others ?


Your insights and kind sharing are heartedly appreciated.

Martin


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