Dear all yes my paalet is large enough and no my protein is not soluble ,and I am using 37degrees before and after induction with IPTG,and the vector that I am using is pRSETA 3.2kd Best Regards Rana
________________________________ From: Kelly Daughtry <kellydaugh...@gmail.com> To: rana ibd <rna19792...@yahoo.com> Cc: CCP4BB@jiscmail.ac.uk Sent: Tuesday, March 27, 2012 5:45 PM Subject: Re: [ccp4bb] dea all When you lyse the cells and spin down cellular debris, is the pellet large and white (indicating inclusion bodies)? Is your protein soluble or membrane? What temperature did you use for expression? What vector are you using? Providing more details allows us to better answer your questions. Off the top of my head: Altering expression can include lowered the temperature just prior to induction (25, 18, or lower) and letting the cells grow overnight. Induction at increased cell density (1.0 vs 0.6 O.D.). Anther option to increase the expression of soluble protein is to use the auto-induction media: http://www.ncbi.nlm.nih.gov/pubmed/15915565 Another option is to try other cells lines, and co-expression with chaperonins (the arctic express cell line is useful http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=467 ). Did you try other tags (GST, MBP, etc)? Hope this helps, Kelly Daughtry ******************************************************* 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 Tue, Mar 27, 2012 at 10:35 AM, rana ibd <rna19792...@yahoo.com> wrote: Dear all >I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB >madia, I am having problems with the expression which shows small amount of >the protein , I also have problems with purification using NI-NTA by also having small amount even after extensive buffer exchange , Is it likely due to the small amount of protein in the medium , should I use a different kind of media, any sugestions or any kind of details or a paper that might help I will be thankful > > >Best Regards >Rana
