Just beware that changing how you break the cells open can change the average size of chromosome chunks, which can change how DNA binding proteins behave in the lysate.
---- Original message ---- >Date: Mon, 3 Mar 2008 15:21:15 +0000 >From: Mads Gabrielsen <[EMAIL PROTECTED]> >Subject: [ccp4bb] finicky protein >To: CCP4BB@JISCMAIL.AC.UK > >I am not a big fan of sonication. Try changing your way of disrupting the >cells. > >I have compared sonication vs mechanical stress on several unrelated proteins, >and for me a good old french press wins every time. If you want to get all >modern and fancy, a cell disruptor gives similar results. > >Cheers, > >Mads Gabrielsen > > >[Hide Quoted Text] > >On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: > >Hi all > >sorry, for offtopic query... > >I am trying to purify my protein by Ni-NTA affinity chromatography. After >sonication as i centrifuge bacterial lysate, soon after 10 min whole >lysates >get precipitated during loading on the column and some time it remain >soluble too. if i get purified through the column without precipitation, >it >gets precipitated during dialysis. >I have tried lot, by chnaging buffers, increasing salt or deacreasing salt >or no salt at are helpless. >I do purifiaction in cold room. > >can any one suggest some solution? > >Thanks in advance. > >NSH > > >-- >Dr. Mads Gabrielsen > >GBRC, B217 >Division of Biochemistry and Molecular Biology >IBLS >University of Glasgow Phone Office: 01413308119 >G12 8QQ Phone Lab: 01413306449 >UK E-mail: [EMAIL PROTECTED] > >------------------------------------------------------------ ---- >This message was sent using IMP, the Internet Messaging Program.
