can you specify what your cell cracker is. Thanks,
Jürgen On Jun 21, 2012, at 9:50 AM, Kelly Daughtry wrote: If it is inclusion bodies, I generally see a milky solution after lysis. Have you centrifuged post-lysis and then run an SDS-PAGE? Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Thu, Jun 21, 2012 at 9:33 AM, RHYS GRINTER <r.grinte...@research.gla.ac.uk<mailto:r.grinte...@research.gla.ac.uk>> wrote: Hi, I often see no real change in change in solution appearance after sonication mediated lysis, with proteins which yield low amounts or no soluble protein in E. coli. I've had a look at the solution post lysis under the microscope and the cells are infact lysed, it's just the presence of high levels of inclusion bodies means the solution remains turbid. Check your pre and post lysis solution under the microscope to see if you see the same thing. Cheers, Rhys ________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of J. Valencia S. [valen...@gene.nagoya-u.ac.jp<mailto:valen...@gene.nagoya-u.ac.jp>] Sent: 21 June 2012 13:44 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Expressed protein hinders cell lysis? Greetings, everyone. We need to ask your advice on an issue with one of our proteins expressed in E. coli Rosetta cells. This yeast-derived protein has a very low yield compared to others we work with, and we think it is because the cells are hard to lyse: even after 3 cycles in a cell cracker the solution barely changes colour. We have no problems lysing Rosetta cells expressing other yeast-derived soluble proteins, and we usually obtain enough for our crystallisation screens. For the aforementioned protein we have already tried using STAR cells, varying the contents of the lysis buffer, sonicating, or adding FeSO4 to the solution (we think the protein binds Fe or Mn because it is yellow), but to no avail. Searching the ccp4bb archive and other resources did not help, so we would like to ask 2 questions to the community in order to focus our efforts better: 1. How can a recombinant protein make a cell harder to lyse? 2. Do you have any suggestions to avoid this effect? We appreciate any input, and will be sure to post a summary for future reference once this issue is solved. Sincerely, -- J. Valencia S. PhD student CGR-NU ...................... 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://web.mac.com/bosch_lab/
