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> 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] On Behalf Of J. > Valencia S. [valen...@gene.nagoya-u.ac.jp] > Sent: 21 June 2012 13:44 > To: 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 >
