0.5% octyl-thioglucopyranoside + lysozyme + DNAse in 30 mM TRIS pH 8.0 - degassed/sparged and supplemented with whatever reducing agent you need. Stir in cold for 30-40 minutes at a ratio of 3ml of solution to 1g of cell pellet (wet).
Artem On Tue, Nov 29, 2011 at 8:49 AM, Jim Spencer, Cellular and Molecular Medicine <[email protected]> wrote: > Dear All, > > Apologies for the off-topic question. I'm seeking suggestions on the best > way to achieve an effective, but not over-harsh, chemical lysis of E coli > expressing an oxygen-sensitive Fe-S protein. We need to lyse anaerobically > but do not have access to a sonicator that we can use in the glove box. We > have used a proprietary detergent mix in previous attempts but have not > been overly impressed with the results- lots of protein (more than I would > expect based on past experiences with related enzymes) remains in the > pellet. I've seen literature protocols based on Triton (up to 1.2%) but am > worried (perhaps without basis) that this might interfere with downstream > steps (reconstituting the Fe-S cluster and possibly crystallisation). The > protein is His-tagged. > > Does anyone know a better way? > > Thanks in advance. > > Best wishes > > Jim > > ---------------------- > Dr. James Spencer, > Lecturer in Microbiology > School of Cellular and Molecular Medicine > Medical Sciences Building > University of Bristol > University Walk > Bristol BS8 1TD > [email protected] > http://www.bristol.ac.uk/**cellmolmed/staff/spencer.html<http://www.bristol.ac.uk/cellmolmed/staff/spencer.html> > ---------------------- > > Tel: (44) (0) 117 331 2084 > Fax: (44) (0) 117 331 2091 >
