Adapted from periplasmic fractionation protocol, but for cytoplasmic
proteins:
1. Incubate resuspended cells with lysozyme in normal buffer but with
20% sucrose.
2. Spin down sphaeroplasts (unbroken cells with no cell wall - ultra
fragile). This also removes periplasmic proteases.
3. Resuspend white pellet in buffer of choice and freeze-thaw. Or
dilute buffer for osmotic shock. Or sonicate *very* lightly -
perhaps using an aging sonicator!
4. Gives complete lysis from 2 ml to 2 litre culture volumes very
reproducibly, and avoids proprietary bug-buster type detergent mixes.
See this paper - it also greatly increases purification (10-fold) of low
abundance his tag proteins that are otherwise outcompeted by periplasmic
components (siderophores?)
http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html
Darren
On 04/02/14 17:49, Phoebe A. Rice wrote:
Some time ago, there was a nice discussion of cost-effective, wimpy
protein-friendly ways to break open E. coli. We're thinking about
replacing an aging sonicator. If people have a favorite gizmo, could
they repeat that advice?
thank you,
Phoebe Rice
++++++++++++++++++++++++++++++++++++++++++
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu <mailto:pr...@uchicago.edu>
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp
--
**********************************************************************
Dr. Darren J. Hart,
CNRS Research Director, Unit of Virus Host Cell Interactions (UVHCI)
Unité Mixte Internationale UMI 3265 (CNRS-EMBL-UJF)
Director, Integrated Structural Biology Grenoble (ISBG)
Unité Mixte de Service UMS 3518 (CNRS-CEA-UJF-EMBL)
**********************************************************************
Email: h...@embl.fr
Tel: +33 4 76 20 77 68; Fax: +33 4 76 20 71 99
Postal address: UVHCI/ISBG, 6 rue Jules Horowitz, BP181, 38042 Grenoble,
Cedex 9, France
For funded access to ESPRIT construct screening via EU FP7 BioStruct-X:
www.biostruct-x.eu/
**********************************************************************
