We do it differently :-) 1 g cell in 2ml of buffer Typically we get anywhere between 10-20 g per liter of TB medium. Since we pass our cells through a cell disruptor and wash afterwards with buffer to maximize our recovery we end up after cell lysis with about 1g in 3-4 ml buffer roughly due to the dilution. We add Benzonase and have 500 mM NaCl in the lysis buffer. I should mention also that the lysis buffer may vary depending on the protein as we go back and fourth optimizing according to our needs.
Try a denaturing purification first and see if anything is expressed (assuming you are His-tagging the protein). Also was the construct sequence confirmed, trivial but not everybody checks the constructs. Jürgen On Oct 25, 2012, at 11:13 AM, Raji Edayathumangalam wrote: Thanks for the confirmation. Raji On Thu, Oct 25, 2012 at 10:44 AM, Rob Gillespie <robert.gilles...@duke.edu<mailto:robert.gilles...@duke.edu>> wrote: Put me down at another person who re-suspends bacterial cell pellets in 4-5 volumes of buffer. On Thu, Oct 25, 2012 at 9:15 AM, Opher Gileadi <opher.gile...@sgc.ox.ac.uk<mailto:opher.gile...@sgc.ox.ac.uk>> wrote: It makes sense to use a fixed ratio of resuspension buffer to cell weight; we weigh the pellets after centrifugation, then suspend in at least 4-5 volumes (ml/gr) of buffer. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
